Transgenic mouse having a transgene that converts a prodrug into a cytotoxic compound in senescent cells

ABSTRACT

This invention provides a transgenic mouse for studying the role of senescent cells on an age-related disorder or an age-sensitive trait. The transgene contains a p16 promoter sequence that controls expression of an enzyme so as to cause the enzyme to be expressed in senescent cells in the mouse. The enzyme converts a prodrug to a cytotoxic agent, so that treating the mouse with the prodrug results in the prodrug selectively killing the senescent cells. As a result, progression of an age-related disorder or an age-sensitive trait is delayed. Included is the 3MR mouse model, which also expresses bioluminescent and fluorescent markers under control of the p16 promoter so that senescent cells in the mice can be visualized.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/692,613 filed Aug. 23, 2012, U.S.Provisional Application No. 61/837,096 filed Jun. 19, 2013, whichapplications are incorporated herein by reference in their entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 200201_410_SEQUENCE_LISTING.txt. The text fileis 31 KB, was created on Aug. 21, 2013, and is being submittedelectronically via EFS-Web.

BACKGROUND

Technical Field

This disclosure relates to non-human animal models for age-relateddisorders and age-sensitive traits associated with senescence-inducingstimuli, methods for identifying therapeutic agents for treating orpreventing age-related disorders and age-sensitive traits associatedwith senescence-inducing stimuli, and methods for treating or preventingage-related disorders and age-sensitive traits. It also relates totherapeutic agents and pharmaceutical compositions for treating orpreventing age-related disorders and age-sensitive traits associatedwith senescence-inducing stimuli.

Description of the Related Art

The American Cancer Society estimates that there will be more than 1.6million new cases of cancer in 2012 in the United States alone. Morethan half of these patients will receive chemotherapy and/or treatmentwith radiation in addition to undergoing surgical resection of thetumor. Various chemotherapeutics and radiation are known to inducecellular senescence. Given that senescent cells have been causallyimplicated in certain aspects of age-related decline in health and maycontribute to certain diseases, the induction of senescent cellsresulting from necessary live-preserving chemotherapeutic and radiationtreatments may have deleterious effects to millions of patientsworldwide (e.g. fatigue, weakness, loss of physical agility). As such,treatments aimed at clearing therapy-induced senescent cells andimproving age-sensitive traits associated with the same have thepotential to markedly improve the health, lifespan and quality of lifefor patients exposed to senescence-inducing stimuli. The presentdisclosure addresses these needs and offers numerous related advantages.

BRIEF SUMMARY

Briefly, provided herein are animal models, methods for identifyingtherapeutic agents, and therapeutic agents useful for treating and/orpreventing age-related disorders and age-sensitive traits, particularlythose cause by senescence-inducing stimuli. For example, provided hereinare the following embodiments.

The present disclosure provides, in one embodiment, a non-human animalmodel for aging comprising a non-human animal that (a) exhibits anage-related disorder or age-sensitive trait and (b) comprises atransgene selectively expressed by senescent cells, wherein the animalis exposed to or treated with a senescence-inducing stimulus. In anotherembodiment, the transgene comprises a senescent cell-specific promoter.In a more specific embodiment, the promoter is derived from p16^(Ink4a).

In another embodiment, the transgene expresses at least one detectablelabel, a cytotoxic agent, a cytotoxicity-activating molecule, an RNA, ora combination thereof. In still another embodiment, the detectable labelis selected from the group consisting of (a) luciferase; (b) a redfluorescent protein; (c) a green fluorescent protein; and (d) aluciferase and a red fluorescent protein. In yet another embodiment, thecytotoxicity-activating molecule is a truncated herpes simplex virusthymidine kinase or a FK506-binding protein (FKBP)-caspase fusionpolypeptide.

In a further embodiment, the detectable label is selected from the groupconsisting of (a) luciferase; (b) a red fluorescent protein; (c) a greenfluorescent protein; and (d) a luciferase and a red fluorescent protein;and wherein the cytotoxicity-activating molecule is a truncated herpessimplex virus thymidine kinase or a FKBP-caspase fusion polypeptide.

In still another embodiment, the transgene comprises (a) a p16^(Ink4a)promoter operatively linked to a polynucleotide sequence encoding aFKBP-caspase fusion polypeptide (p16-FKBP-caspase transgene), and to apolynucleotide sequence encoding a green fluorescence protein, or (b) ap16^(Ink4a) promoter operatively linked to a polynucleotide sequenceencoding a fusion polypeptide comprising a luciferase, a red fluorescentprotein, and a truncated herpes simplex virus thymidine kinase (p16-3MRtransgene).

In yet another embodiment, the transgene comprises a p16^(Ink4a)promoter operatively linked to a polynucleotide sequence encoding aFKBP-caspase fusion polypeptide (p16-FKBP-caspase transgene), and to apolynucleotide sequence encoding a green fluorescence protein.

In a further embodiment, the transgene comprises a p16^(Ink4a) promoteroperatively linked to a polynucleotide sequence encoding a fusionpolypeptide comprising a luciferase, a red fluorescent protein, and atruncated herpes simplex virus thymidine kinase (p16-3MR transgene).

In another embodiment, the senescence-inducing stimulus gives rise to anage-related disorder or age-sensitive trait. In a more specificembodiment, the senescence-inducing stimulus comprises exposure toirradiation or treatment with a chemotherapeutic agent. In anotherspecific embodiment, the chemotherapeutic agent is doxorubicin, taxol,docetaxel, gemcitabine, or cisplatin. In yet another specificembodiment, the irradiation is whole body gamma radiation.

In still another embodiment, the age-related disorder or age-sensitivetrait results at least in part from (1) a genetic modification; (2) adiet modification; (3) a chemical induction; (4) radiation induction; or(5) any combination thereof. For example, in a more specific embodiment,the age-related disorder or age-sensitive trait results at least in partfrom a genetic modification, wherein the genetic modification comprises(1) expression of a second transgene; (2) reduced or abrogatedexpression of an endogenous gene, or (3) a combination thereof. Inanother specific embodiment, the animal is a BubR1 mouse.

The present disclosure provides, in another embodiment, a non-humananimal model comprising a transgene that comprises (1) a senescentcell-specific promoter operatively linked to a polynucleotide encoding(a) at least one detectable label, (b) a cytotoxic agent, (c) acytotoxicity-activating molecule, (d) an RNA, or (e) any combination of(a), (b), (c), and (d); and wherein the animal exhibits an age-sensitivetrait. In another embodiment, the transgene comprises a p16^(Ink4a)promoter operatively linked to a polynucleotide sequence encoding aFKBP-caspase fusion polypeptide (p16-FKBP-caspase transgene) and a greenfluorescent protein. In still another embodiment, the transgenecomprises a p16^(Ink4a) promoter operatively linked to a polynucleotidesequence encoding a fusion polypeptide comprising a luciferase, a redfluorescent protein, and a truncated herpes simplex virus thymidinekinase (p16-3MR transgene).

In yet another embodiment, the age-sensitive trait is measure of aspecific T cell subset distribution, cataract formation, spontaneousactivity, motor coordination, cognitive capacity, physical function,body composition, cardiac function, or any combination thereof. In amore specific embodiment, the body composition is a measure ofsarcopenia, osteoporosis, bone mineral density, lean mass or fat mass.In a related embodiment, the lean mass, fat mass and bone mineraldensity are measured by QNMR, dual-energy X-ray absorptiometry, MRI,PET, or a combination thereof. In another related embodiment, thephysical function is a measure of (1) running time, distance, and workusing a motorized treadmill, (2) grip strength using a grip meter, or(3) any combination measure thereof. In another embodiment, theage-sensitive trait is a measure of a tissue or organ, wherein themeasure is of fiber diameter on gastrocnemius muscle, DNA damage, renaland glomerulosclerosis, retinal atrophy, proteotoxic stress, oxidativestress, or hematopoietic system.

The present disclosure provides, in another embodiment, a method foridentifying a therapeutic agent effective for treating or reducing therisk of developing an age-related disorder or age-sensitive trait, saidmethod comprising:

(a) administering a candidate therapeutic agent to the animal of theaging animal model of the present disclosure, to provide a treatedanimal;

(b) (1) determining an age-related disorder or age-sensitive traitexhibited in the treated animal, and comparing to the age-relateddisorder or age-sensitive trait exhibited in an untreated control agingmodel animal, or

(2) determining the level of suppression of cellular senescence in thetreated animal and comparing to the level of cellular senescence in theuntreated control animal

wherein (1) suppression of an age-related disorder or age-sensitivetrait or (2) suppression of cellular senescence in the treated animalcompared with the untreated animal identifies an agent effective fortreating or preventing an age-related disorder or age-sensitive trait.

In a more specific embodiment, step (b) comprises:

(1) determining an age-related disorder or age-sensitive trait exhibitedin the treated animal, and comparing to the age-related disorder orage-sensitive trait exhibited in an untreated control aging modelanimal; and

(2) determining the level of suppression of cellular senescence in thetreated animal and comparing to the level of cellular senescence in theuntreated control animal; and

wherein (1) suppression of an age-related disorder or age-sensitivetrait and (2) suppression of cellular senescence in the treated animalcompared with the untreated animal identifies an agent effective fortreating or preventing an age-related disorder or age-sensitive trait

In a related embodiment, suppression of cellular senescence comprisessuppression of the expression or secretion of one or more senescentcell-associated molecules in the animal. In yet another relatedembodiment, suppression of cellular senescence comprises reducing thequantity of senescent cells in the animal.

The present disclosure provides, in another embodiment, a therapeuticagent for treating or reducing the likelihood of developing anage-related disorder or age-sensitive trait, wherein the therapeuticagent is identified according to a method of the present disclosure.

The present disclosure provides, in another embodiment, a method fortreating or reducing the likelihood of developing an age-relateddisorder or age-sensitive trait in a subject who has an age-relateddisorder or age-sensitive trait, who is in remission for an age-relateddisorder or age-sensitive trait, or who is at risk of developing arecurrence of an age-related disorder or age-sensitive trait, comprisingadministering to the subject the therapeutic agent of the presentdisclosure.

The present disclosure also provides, in another embodiment, a methodfor treating or reducing the likelihood of an age-related disorder orage-sensitive trait in a subject who has an age-related disorder orage-sensitive trait, who is in remission for an age-related disorder orage-sensitive trait, or who is at risk of developing a recurrence of anage-related disorder or age-sensitive trait, said method comprisingadministering a therapeutic agent that selectively suppresses cellularsenescence in the subject, thereby treating or reducing the likelihoodof an age-related disorder or age-sensitive trait in the subject. Inanother embodiment, suppressing cellular senescence comprisessuppressing the expression or secretion of one or more senescentcell-associated molecules in the subject. In a related embodiment,suppressing cellular senescence comprises reducing the quantity ofsenescent cells in the subject.

The present disclosure also provides, in another embodiment, an isolatedcell or cell line derived from the animal model of the presentdisclosure.

The present disclosure further provides, in another embodiment, a methodfor producing the non-human animal of the model of the presentdisclosure, comprising (a) providing a non-human animal that comprises atransgene selectively expressed by senescent cells; and (b) aging theanimal to produce an age-related disorder or age-sensitive trait.

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments.However, one skilled in the art will understand that the invention maybe practiced without these details. In other instances, well-knownstructures have not been shown or described in detail to avoidunnecessarily obscuring descriptions of the embodiments. Unless thecontext requires otherwise, throughout the specification and claimswhich follow, the word “comprise” and variations thereof, such as,“comprises” and “comprising” are to be construed in an open, inclusivesense, that is, as “including, but not limited to.” In addition, theterm “comprising” (and related terms such as “comprise” or “comprises”or “having” or “including”) is not intended to exclude that in othercertain embodiments, for example, an embodiment of any composition ofmatter, composition, method, or process, or the like, described herein,may “consist of” or “consist essentially of” the described features.Headings provided herein are for convenience only and do not interpretthe scope or meaning of the claimed embodiments.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

Also, as used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “anon-human animal” may refer to one or more non-human animals, or aplurality of such animals, and reference to “a cell” or “the cell”includes reference to one or more cells and equivalents thereof (e.g.,plurality of cells) known to those skilled in the art, and so forth.When steps of a method are described or claimed, and the steps aredescribed as occurring in a particular order, the description of a firststep occurring (or being performed) “prior to” (i.e., before) a secondstep has the same meaning if rewritten to state that the second stepoccurs (or is performed) “subsequent” to the first step. The term“about” when referring to a number or a numerical range means that thenumber or numerical range referred to is an approximation withinexperimental variability (or within statistical experimental error), andthus the number or numerical range may vary between 1% and 15% of thestated number or numerical range. It should also be noted that the term“or” is generally employed in its sense including “and/or” unless thecontent clearly dictates otherwise. The term, “at least one,” forexample, when referring to at least one compound or to at least onecomposition, has the same meaning and understanding as the term, “one ormore.”

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D include a listing of an exemplary transgene selectivelyexpressed in senescent cells, the nucleic acid sequence of a pBLUESCRIPTII KS vector containing a p16^(Ink4a) promoter-FKBP-caspase 8-IRES-GFPnucleic acid construct.

FIGS. 2A-2F include a listing of the nucleic acid sequence of FIG. 1with the various vector components and construct components labeled.

FIG. 3 represents an exemplary 3MR transgene sequence.

FIG. 4 illustrates lifespan difference between wild-type INK-ATTACtransgenic mice treated with AP20187 (TG TR) and untreated wild-typeINK-ATTAC transgenic mice (TG UN).

FIG. 5 shows fat mass measurements for 18-month wild-type INK-ATTACtransgenic mice treated with AP20187 or control INK-ATTAC animals(treated with PBS). Treated female mice were compared with untreatedfemale mice, and treated male mice were compared with untreated malemice.

FIG. 6 illustrates the difference in adipose cell (AC) diameter inAP20187-treated wild-type INK-ATTAC transgenic mice (TG3+/TG5+,indicating mice from two different INK-ATTAC strains) and controluntreated (PBS) INK-ATTAC animals (TG3−).

FIG. 7 illustrates cardiac performance of AP20187-treated wild-typeINK-ATTAC transgenic mice (treated) and untreated (PBS) INK-ATTAC mice.Animals were injected with isoproterenol, as an exogenous stress, priorto sacrifice of the animals.

FIG. 8 shows the extent of glomerulosclerosis in AP20187-treatedwild-type INK-ATTAC transgenic mice (TG+) and untreated (PBS) wild-typeINK-ATTAC mice.

DETAILED DESCRIPTION

The present disclosure relates generally to non-human animal models ofaging. More specifically, the present disclosure provides non-humananimal models of aging, age-related disorders, age-sensitive traits, andthe like, wherein the animals models comprise transgenes selectivelyexpressed by senescent cells, and wherein the age-related disorders andage-sensitive traits result at least in part from senescence-inducingstimuli, such as treatment with chemotherapy or radiation. For example,in certain embodiments, the animal models comprise a transgene thatallows for the selective and controllable ablation of senescent cells ata desired time or for a desired period relative to exposure of theanimals to senescence-inducing stimuli. The animal models areadvantageously used for determining the deleterious health effects ofsenescence-inducing stimuli and for identifying therapeutic agentseffective for treating or preventing age-related disorders andage-sensitive traits associated with or caused by such stimuli. Asdescribed herein, the present disclosure also provides therapeuticagents identified using such methods, pharmaceutical compositionscomprising the identified therapeutic agents, and methods of treating orpreventing age-related disorders and age-specific traits associated withsenescence-inducing stimuli.

Transgenic Animals with Senescent Cell-Specific Transgene Expression

Certain aspects of the present disclosure employ non-human animals,particularly genetically modified non-human animals, wherein the animalscomprise a transgene expressed under the control of a senescentcell-specific promoter, and wherein the animals are exposed to ortreated with at least one senescence-inducing stimulus, such aschemotherapy or radiation. In certain more specific embodiments, theanimals comprise a transgene that allows for the selective andcontrollable ablation of senescent cells.

By operably linking a senescent cell-specific promoter of a transgene toa nucleic acid sequence encoding a polypeptide of interest (e.g., adetectable label or cytotoxicity-activating molecule), senescent cellswithin an animal can be monitored and/or deleted in a controlled anduser-determined fashion. In certain embodiments, for example, thepresent disclosure employs transgenic non-human animals that can beinduced to delete senescent cells in vivo at a predetermined and desiredpoint in time, such as at a particular stage of development or disease.

In addition to the transgene selectively expressed by senescent cells,an animal model of the present disclosure, to which asenescence-inducing stimulus is administered, may further contain othergenetic or non-genetic modifications, or may be exposed to oradministered other agents or treatments that are known to accelerate orfacilitate the aging process in the animal and/or that contribute to thedevelopment of an age-related disorder or age-sensitive trait in theanimal. For example, as further described herein, the non-human animalscomprising a senescent cell-specific transgene may be advantageouslycrossed with other animals, which, due to one or more geneticmodifications, for example, are known to develop a desired age-relatedphenotype, age-related disorder and/or age-sensitive trait of interest.

In this way, an animal model may be generated according to the presentdisclosure which is genetically or otherwise modified to develop adesired age-related phenotype, age-related disorder and/or age-sensitivetrait, wherein the role of senescence-inducing stimuli in relation to ananimal's age-related phenotype, age-related disorder and/orage-sensitive trait may be evaluated. The animal model may be furtheremployed in screening methods in order to identify therapeutic agentthat suppress or otherwise advantageously effect senescent cellsurvival, viability and/or clearance in the context of the animal model.

A senescent cell-specific promoter sequence present within a transgeneof the present disclosure can be essentially any sequence thatselectively drives expression of a polypeptide encoded by the transgeneor expression of a nucleic acid sequence (e.g., an RNA) in senescentcells, while driving less, little, or no expression of the encodedpolypeptide or nucleic acid sequence in non-senescent cells. In certainexemplary embodiments, a senescent cell-specific promoter used inaccordance with the present disclosure may include, without limitation,a p16^(Ink4a) promoter sequence, a p21cip promoter sequence, or a Pailpromoter sequence.

In certain embodiments, the present disclosure provides a non-humananimal model comprising a transgene that comprises (1) a senescentcell-specific promoter operatively linked to a polynucleotide encoding(a) at least one detectable label, (b) a cytotoxic agent, (c) acytotoxicity-activating molecule, (d) an RNA, or (e) any combination of(a), (b), (c) and (d); and that exhibits a phenotype of an age-relateddisease.

It will be understood that a senescent cell-specific promoter can beoperably linked to a nucleic acid sequence encoding any polypeptide ofinterest. In certain embodiments, the polypeptide of interest isselected from a detectable label, a cytotoxic molecule (e.g., apolypeptide capable of killing a senescent cell in which it isexpressed) and a cytotoxicity-activating molecule (e.g., a polypeptidecapable of facilitating the killing of a cell in which it is expressed),or a combination thereof. In certain embodiments, the transgene can beoperably linked to an RNA (e.g., siRNA, shRNA, microRNA, and the like,which reduces or abrogates the expression of one or more genes importantor essential for senescent cell survival), or a combination thereof.

Depending on the polypeptide of interest that is encoded by thetransgene, it will be understood that different promoter features may beadvantageous or desired. For example, as will be understood, inembodiments wherein a transgene encodes a polypeptide that is directlycytotoxic to cells in which it is expressed, the senescent cell-specificpromoter of the transgene will need to be an inducible promoter in orderto control the timing of expression of the cytotoxic polypeptide, andthereby control the deletion of senescent cells in the animal. If, onthe other hand, a transgene encodes a cytotoxicity-activatingpolypeptide, the transgene promoter need not be inducible. Rather, theinducibility of the system in this instance relies upon the timing ofexposure of the cytotoxicity-activating molecule expressed by senescentcells to its activating agent, as further described below.

Any of a number of detectable labels may be operably linked to asenescent cell-specific promoter. Many such detectable labels, and themeans by which they can be detected, have been described and are wellknown and established in the art. In some embodiments, the detectablelabel comprises one or more fluorescent or bioluminescent labels, manyof which are well known and established in the art. For example, thefluorescent protein may be any protein that fluoresces and that may bevisualized when expressed in senescent cells under the control of asenescent cell-specific promoter as described herein. Illustrativefluorescent proteins can include, for example, green fluorescent protein(GFP), modified or enhanced green fluorescent protein (EGFP), redfluorescent protein (RFP), and various other known fluorescent proteinssuch as EBFP, EBFP2, Azurite, mKalama1, ECFP, Cerulean, CyPet, YFP,Citrin, Venus, and Wet. Other illustrative fluorescent or bioluminescentproteins include, for example, infrared-fluorescent proteins (IFPs),mRFP1, mCherry, mOrange, DsRed, tdTomato, mKO, TagRFP, mOrange2, maple,TagRFP-T, Firefly Luciferase, Renilla Luciferase and Click BeetleLuciferase. Still other illustrative labels can include yellowfluorescent protein, cyan FP, blue FP, red FP and their enhancedversions. It will be understood that essentially any of a number ofother luminescent or fluorescent proteins that can emit light can beused in this context.

In certain specific embodiments, a detectable label present within atransgene according to the disclosure is selected from the groupconsisting of (a) luciferase; (b) a red fluorescent protein; (c) a greenfluorescent protein; and (d) a luciferase and a red fluorescent protein.

Any of a number of cytotoxicity-activating molecules may be operablylinked to a senescent cell-specific promoter to produce a suitabletransgene for use in the context of the present disclosure. Followingits expression in a senescent cell-specific fashion, thecytotoxicity-activating molecule is one that is capable of inducing thecontrollable killing of the senescent cells in which it is expressedupon administration of an activating agent to the transgenic animal.Illustrative examples of cytotoxicity-activating molecules include, butare not limited to herpes simplex virus (HSV) thymidine kinase (TK)polypeptides and FK506 binding protein (FKBP)-caspase fusionpolypeptide. FK506 binding protein includes variant thereof, such as aPhe36Val mutant.

For example, in a specific embodiment, the cytotoxicity-activatingmolecule encoded by the transgene is a herpes simplex virus (HSV)thymidine kinase (TK) polypeptide (including truncated TK polypeptides)and the activating agent is ganciclovir.

In other embodiments, the cytotoxicity-activating molecule encoded bythe transgene comprises two or more polypeptide sequences fused together(e.g., a fusion polypeptide). An example of such a fusion polypeptidecan be a FKBP-caspase-8 fusion polypeptide. See, e.g., Pajvani et al.,Nat. Med., 11:797-803 (2005) and Baker et al., Nature 479:232-36 (2011).Such fusion polypeptides may comprise, for example, one or morecatalytic domains of human caspase-8 fused to one or more FKBP domains.Following transgene expression, adjacent FKBP molecules in the encodedpolypeptide can be activated via forced dimerization using a suitableactivating agent, thereby allowing for the regulated ablation of cellsin which the fusion polypeptide is expressed. In a specific example, thep20 and p10 domains of human caspase-8 are fused to serial FKBPv(Phe36Val mutant FKBP) domains. Other examples of such polypeptidesinclude, without limitation, a FKBP-caspase-1 fusion polypeptide orFKBP-caspase-3 fusion polypeptide (see, e.g., Mallet et al., Nat.Biotechnol. 20:1234-39 (2002)). In these and related embodiments, anillustrative activating agent used to induce cytotoxicity of senescentcells expressing the fusion polypeptide include the compound FK1012analog AP20187 (referred to herein as AP20187) and related analogs.(see, e.g., U.S. Patent Application Publication No. 2004/0006233, thedisclosure of which is incorporated herein by reference). To increaselocal concentrations of a FKBP-caspase fusion polypeptide, amyristoylation sequence may be included in the transgene to providemembrane attachment for the FKBP-caspase fusion polypeptide.

In this way, administration of a suitable cytotoxicity activatingmolecule to an animal at a desired time provides an effective means forselectively killing (e.g., by apoptosis) the senescent cells whichexpress the cytotoxicity-activating molecule in the animal. In certainspecific embodiments, less than 1%, 5%, 10% or 20% of non-senescentcells of the transgenic mouse are killed when an activating compound isadministered to a transgenic mouse comprising a transgene encoding acytotoxicity activating molecule.

Any of a number of nucleotide sequences encoding small RNAs whoseexpression affects expression or secretion of senescence cell-associatedmolecules may be operatively linked to a senescent cell-specificpromoter. Such small RNAs include siRNA, shRNA, microRNA and the like(see, Finnegan and Matzke, J. Cell Sci. 226:4689-93, 2003). In certainembodiments, the expression of such small RNAs is under the control ofan inducible senescent cell-specific promoter. Upon induction, theexpression of the small RNAs down-regulates the expression or secretionof senescent cell-associated molecules of interest.

In some cases, a polypeptide encoded by a transgene of the presentdisclosure will be engineered to include one or more other elements,such as an affinity tag (e.g., a Flag tag), cellular localizationsequence (e.g., myristoylation sequence) or any other desired element ofinterest.

In light of the above general disclosure, it will be evident thatvarious more specific transgenes and transgenic non-human animals areprovided for use herein. For example, in a specific embodiment, theanimal model will comprise a transgene wherein a senescent cell-specificpromoter directs expression of a detectable label selected from thegroup consisting of (a) luciferase; (b) a red fluorescent protein; (c) agreen fluorescent protein; and (d) a luciferase and a red fluorescentprotein; and further directs expression of a cytotoxicity-activatingmolecule selected from the group consisting of a truncated herpessimplex virus thymidine kinase and a FKBP-caspase fusion polypeptide.

In another specific embodiment, an animal model includes a transgenecomprising (a) a p16^(Ink4a) promoter operatively linked to apolynucleotide sequence encoding a FKBP-caspase fusion polypeptide(p16-FKBP-caspase transgene) and to a polynucleotide sequence encoding agreen fluorescent protein; or (b) a p16^(Ink4a) promoter operativelylinked to a polynucleotide sequence encoding a fusion polypeptidecomprising a luciferase, a red fluorescent protein, and a truncatedherpes simplex virus thymidine kinase (tTK) (p16-3MR transgene), whichmay be called herein a trimodal fusion protein (3MR). In more specificembodiments, luciferase is renilla luciferase and red fluorescentprotein is a monomeric red fluorescent protein. A p16^(Ink4a) promotermay comprise a full-length promoter sequence or may comprise afunctional (i.e., operable) truncation (or fragment) thereof (see, e.g.,Wang et al. J. Biol. Chem. 276:48655-61 (2001)).

The non-human animal models of the present disclosure can be implementedin essentially any type of animal. Most typically, the animal model willbe a mammal. In more specific embodiments, illustrative animal modelsinclude, but are not limited to, models derived from farm animals suchas pigs, goats, sheep, cows, horses, and rabbits, rodents such as rats,guinea pigs, and mice, and non-human primates such as baboons, monkeys,and chimpanzees.

Transgenic nonhuman animal of the present disclosure can include,without limitation, founder transgenic non-human animals as well asprogeny of the founders, progeny of the progeny, and so forth, providedthat the progeny retain the transgene. The nucleated cells of thetransgenic nonhuman animals provided herein can contain a transgene thatincludes a senescent cell-specific promoter sequence (e.g., ap16^(Ink4a) promoter sequence, or an operable truncation thereof)operably linked to a nucleic acid sequence encoding a polypeptide ofinterest, such as a polypeptide that comprises a detectable label, apolypeptide capable of killing a cell (e.g., a cytotoxic polypeptide)and/or a polypeptide capable of facilitating the killing of a cell(e.g., a cytotoxicity-activating polypeptide), or a combination thereof.

In the context of transgenic animal production, operably linking apromoter sequence of interest to a nucleic acid sequence encoding apolypeptide of interest is well known and established. This generallyinvolves positioning a regulatory element (e.g., a promoter sequence, aninducible element and/or an enhancer sequence) relative to a nucleicacid sequence encoding a polypeptide in such a way as to permit orfacilitate expression of the encoded polypeptide. In the transgenesdisclosed herein, for example, a promoter sequence (e.g., a p16^(Ink4a)promoter sequence, or an operable truncation thereof) can be positioned5′ relative to a nucleic acid encoding a polypeptide of interest (e.g.,an FKBP-caspase-8 fusion protein).

Various techniques known in the art can be used to introduce transgenesinto non-human animals to produce founder lines, in which the transgeneis integrated into the genome. Such techniques include, withoutlimitation, pronuclear microinjection (See, e.g., U.S. Pat. No.4,873,191), retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci. USA, 82:6148-1652 (1985)), genetargeting into embryonic stem cells (Thompson et al., Cell 56:313-321(1989)), electroporation of embryos (Lo, Mol. Cell. Biol., 3:1803-1814(1983)), and in vitro transformation of somatic cells, such as cumulusor mammary cells, followed by nuclear transplantation (Wilmut et al.,Nature, 385:810-813 (1997); and Wakayama et al., Nature, 394:369-374(1998)). For example, fetal fibroblasts can be genetically modified tocontain a desired transgene construct, and then fused with enucleatedoocytes. After activation of the oocytes, the eggs are cultured to theblastocyst stage. See, for example, Cibelli et al., Science,280:1256-1258 (1998). Standard breeding techniques can be used to createanimals that are homozygous for the transgene from the initialheterozygous founder animals. Homozygosity is not required, however, asthe phenotype can be observed in hemizygotic animals.

Once transgenic non-human animals have been generated, expression of anencoded polypeptide can be assessed using standard techniques. Initialscreening can be accomplished by Southern blot analysis to determinewhether or not integration of the transgene has taken place. For adescription of Southern analysis, see sections 9.37-9.52 of Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, second edition, ColdSpring Harbor Press, Plainview; NY. Polymerase chain reaction (PCR)techniques also can be used in the initial screening. PCR refers to aprocedure or technique in which target nucleic acids are amplified.Generally, sequence information from the ends of the region of interestor beyond is employed to design oligonucleotide primers that areidentical or similar in sequence to opposite strands of the template tobe amplified. PCR can be used to amplify specific sequences from DNA aswell as RNA, including sequences from total genomic DNA or totalcellular RNA. Primers typically are 14 to 40 nucleotides in length, butcan range from 10 nucleotides to hundreds of nucleotides in length. PCRis described in, for example PCR Primer: A Laboratory Manual, ed.Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.Nucleic acids also can be amplified by ligase chain reaction, stranddisplacement amplification, self-sustained sequence replication, ornucleic acid sequence-based amplified. See, for example, Lewis, GeneticEngineering News, 12:1 (1992); Guatelli et al., Proc. Natl. Acad. Sci.USA, 87:1874-1878 (1990); and Weiss, Science, 254:1292-1293 (1991).

Expression of a nucleic acid sequence encoding a polypeptide of interestin senescent cells of transgenic non-human animals can be assessed usingtechniques that include, without limitation, Northern blot analysis oftissue samples obtained from the animal, in situ hybridization analysis,Western analysis (immunoblot analysis), immunoassays such asenzyme-linked immunosorbent assays, and reverse-transcriptase PCR(RT-PCR).

It will be understood that the present disclosure also provides tissues(e.g., skin, eye, fat, muscle, lung, heart, bone, liver, intestine,kidney, spleen, brain, cartilage, marrow, adrenal glands, ovaries, andtestes) and cells or cell lines (e.g., fat cells, preadipocytes, skin orlung fibroblasts, muscle satellite cells, osteoblasts, bone marrowprogenitor cells, neuronal progenitor cells, hepatocytes, endothelialcells, chondroblasts, and splenocytes cells) obtained from a transgenicnonhuman animal provided herein.

Polypeptide sequences and the encoding polynucleotide sequences forproteins, protein domains and fragments thereof, for proteins describedherein such as HSV thymidine kinase (TK) polypeptides, FK506 bindingprotein (FKBP) and domains thereof, caspase(s) and domains thereof, thedetectable fluorescent, bioluminescent polypeptides that are describedherein include natural and recombinantly engineered variants. Thesevariants retain the function and biological activity (includingenzymatic activities if applicable) associated with the parent (orwildtype) protein. Conservative substitutions of amino acids are wellknown and may occur naturally in the polypeptide (e.g., naturallyoccurring genetic variants) or may be introduced when the polypeptide isrecombinantly produced. Amino acid substitutions, deletions, andadditions may be introduced into a polypeptide using well-known androutinely practiced mutagenesis methods (see, e.g., Sambrook et al.Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring HarborLaboratory Press, N Y 2001)). Oligonucleotide-directed site-specific (orsegment specific) mutagenesis procedures may be employed to provide analtered polynucleotide that has particular codons altered according tothe substitution, deletion, or insertion desired. Deletion or truncationvariants of proteins may also be constructed by using convenientrestriction endonuclease sites adjacent to the desired deletion.Alternatively, random mutagenesis techniques, such as alanine scanningmutagenesis, error prone polymerase chain reaction mutagenesis, andoligonucleotide-directed mutagenesis may be used to prepare polypeptidevariants (see, e.g., Sambrook et al., supra).

Differences between a wild type (or parent) polynucleotide orpolypeptide and the variant thereof, may be determined by methodsroutinely practiced in the art to determine identity, which are designedto give the greatest match between the sequences tested. Methods todetermine sequence identity can be applied from publicly availablecomputer programs. Computer program methods to determine identitybetween two sequences include, for example, BLASTP, BLASTN (Altschul, S.F. et al., J. Mol. Biol. 215: 403-410 (1990), and FASTA (Pearson andLipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988). The BLAST familyof programs is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.

Assays for determining whether a polypeptide variant folds into aconformation comparable to the non-variant polypeptide or fragmentinclude, for example, the ability of the protein to react with mono- orpolyclonal antibodies that are specific for native or unfolded epitopes,the retention of ligand-binding functions, the retention of enzymaticactivity (if applicable), and the sensitivity or resistance of themutant protein to digestion with proteases (see Sambrook et al., supra).Polypeptides, variants and fragments thereof, can be prepared withoutaltering a biological activity of the resulting protein molecule (i.e.,without altering one or more functional activities in a statisticallysignificant or biologically significant manner). For example, suchsubstitutions are generally made by interchanging an amino acid withanother amino acid that is included within the same group, such as thegroup of polar residues, charged residues, hydrophobic residues, and/orsmall residues, and the like. The effect of any amino acid substitutionmay be determined empirically merely by testing the resulting modifiedprotein for the ability to function in a biological assay, or to bind toa cognate ligand or target molecule.

Non-Human Animals Models for Aging, Age-Related Disorders andAge-Sensitive Traits Caused by Senescence-Inducing Stimuli

The present disclosure provides transgenic non-human animal models ofaging, which exhibit, for example, age-related phenotypes, age-relateddisorders, age-sensitive traits, and the like, wherein the animalscomprise a transgene that allows for the selective and controllableablation of senescent cells. The animal models are particularlyadvantageous for determining the contribution of senescence-inducingstimuli to age-related health parameters and for treating thedeleterious effects that result from such stimuli.

A non-human animal model of the disclosure exhibits features of anage-related phenotype, age-related disorder or age-sensitive trait atleast in part as a result of contact with a senescence-inducingstimulus. Exemplary features of an age-related phenotype, age-relateddisorder or age-sensitive trait will be apparent in light of the presentdisclosure.

In certain specific embodiments, for example, an age-sensitive traitevaluated in the context of the animal models is selected from one ormore of T cell subset distribution, cataract formation, spontaneousactivity, motor coordination and cognitive capacity, physical function,body composition (e.g., sarcopenia, osteoporosis, loss of fat mass,adipose tissue loss, adipocyte cell size) and cardiac function. Stillother illustrative age-sensitive traits can be measured using tissuesand organs of test and control mice, including fiber diameter analysison muscle (e.g., gastrocnemius muscle, abdominal muscle), DNA damageanalysis, analysis of renal and glomerulosclerosis, analysis for retinalatrophy, proteotoxic stress analysis, oxidative stress analysis,analysis of the hematopoietic system, and the like.

These exemplary age-sensitive traits can be assessed using standardtechniques known and available in the art. Spontaneous activity ofindividual mice can be measured, for example, over a 48-hour periodusing comprehensive laboratory animal monitoring systems equipped withphotocells (e.g., Columbus Instruments) as previously described (e.g.,Handschin et al., J. Biol. Chem., Vol. 282, 41, 30014, Oct. 12, 2007;Pack et al., Physiol. Genomics (Sep. 19, 2006)). Motor coordination canbe analyzed, for example, by performing an accelerating rotarod test.For measuring cognitive capacity, a modified Stone T-maze, which issensitive to age-related changes in learning and memory, can be used.Physical function can be assessed, for example, by measuring runningtime, distance and work using a motorized treadmill, and grip strengthusing a grip meter, according to previously described protocols (e.g.,Zhang et al., Animal Models of Inflammatory Pain Neuromethods, Volume49, Oct. 20, 2010, 23-40; Balkaya et al., Behavioral Testing in MouseModels of Stroke, Neuromethods, Volume 47, 2010, 179-197). Lean mass,fat mass and bone mineral density can be assessed, for example, by QNMRand/or dual-energy X-ray absorptiometry measurements as previouslydescribed (e.g., Reed et al., Physiology & Behavior, Vol. 91, 2007,593-600; Halldorsdottir et al., Int. J. Body Compos. Res., 2009; 7(4),147-154; Brommage et al., AJP-Endo, Sep. 1, 2003, Vol. 285, No. 3E454-E459).

These and other methods for detecting, monitoring or quantifyingage-related phenotypes associated with senescence-inducing stimuli willbe apparent in light of the present disclosure, such as histologicalstudies, molecular studies, biochemical studies, cognitive studies,behavioral assessment, and others. Exemplary methods are also providedin the examples of the present disclosure.

A senescence-inducing stimulus may include, for example, one or more ofa chemical stimulus, an environmental stimulus, a genetic modification,a diet modification, or a combination thereof. In certain specificembodiments, the senescence-inducing stimulus comprises irradiationtreatment or treatment with one or more chemotherapeutic agents. Incertain more specific embodiments, the chemotherapeutic agents areillustratively selected from doxorubicin, taxol, docetaxel, gemcitabine,or cisplatin. In other embodiments, the senescence-inducing stimulus iscigarette smoking or exposure to cigarette smoke or other environmentalinsults.

An animal comprising a transgene selectively expressed in senescentcells may be exposed to one or more senescence-inducing stimuli (e.g.,radiation or chemotherapy) at any desired points in time.

The aging process in an animal model, or the induction of an age-relateddisorder or age-sensitive trait, can be manipulated by any desiredmeans. For example, a non-human animal model as described herein may bea multi-transgenic model comprising, in addition to a first transgeneselectively expressed in senescent cells, a second transgene whichexpressed a gene product that contributes to a desired age-relatedphenotype (i.e., a transgenic aging model) or having reduced orabrogated expression of an endogenous gene (i.e., gene knockout agingmodel), or a combination thereof, which results in or contributes to anage-related phenotype, including an age-related disorder orage-sensitive trait, in the animal. Alternatively, a non-human animalmodel as described herein may contain one or more naturally occurringmutations that result in or contribute to an age-related phenotype ofinterest. As such, the effects of senescence-inducing stimuli on theage-related phenotype of the animals can be advantageously determined.

In one embodiment, to create an animal model as described herein, atransgenic parent animal comprising a transgene selectively expressed bysenescent cell (for convenience, referred to as a “first transgenicanimal”) and a genetically modified model parent animal, e.g., whichdevelops a desired age-related phenotype (for convenience, referred toas a “second genetically modified parent animal”) may be crossed (orbred) with each other. A transgenic animal comprising a transgeneselectively expressed by a senescent cell may be any transgenic animalthat comprises a senescent cell-specific promoter within a transgenethat selectively drives expression of a polypeptide encoded by thetransgene in senescent cells. In certain embodiments, a first transgenicparent animal comprises a transgene comprising p16^(Ink4a) promoteroperatively linked to a polynucleotide sequence encoding a FKBP-caspasefusion polypeptide (p16-FKBP-caspase transgene), and to a polynucleotidesequence encoding a green fluorescent protein; or a p16^(Ink4a) promoteroperatively linked to a polynucleotide sequence encoding a fusionpolypeptide comprising a luciferase, a red fluorescent protein, and atruncated herpes simplex virus thymidine kinase (p16-3MR transgene). Inother embodiments, a parent animal comprises a transgene comprising ap16^(Ink4a) promoter in frame to express an at least one FKBP domain andat least one caspase domain (e.g., FKBP-caspase fusion polypeptidedescribed herein) and comprises the p16-3MR transgene described herein.In still another embodiment, a parent animal comprises a transgenecomprising a p16^(Ink4a) promoter in frame to express a at least oneFKBP domain and at least one caspase domain (e.g., FKBP-caspase fusionpolypeptide described herein) or the transgene comprises the p16-3MRtransgene described herein, and which parent animal has a BubR1hypomorphic (BubR1H/H) genetic background (see, e.g., Baker et al.,Nature 479:232-236 (2011); International Application Publication No. WO2012/177927). The second genetically modified parent animal may be ananimal that develops an age-related phenotype (e.g., transgenic orknockout models that develop an age-related phenotype). F1 progeny froma cross between these parental animals are multi-transgenic animalswhich express the transgenes from each parent. Additional crosses forselection of progeny with heterozygous or homozygous knockout genes maybe necessary.

In an alternative embodiment, a multi-transgenic model may be derived bydirectly introducing a transgene into the germline of anothergenetically modified animal. For example, a transgene selectivelyexpressed by senescent cells (e.g., p16-3MR transgene) may be injectedinto single cell embryos harvested from a genetically modifiedtransgenic mouse model. A person having skill in the art will understandthat various injection/recipient embryo combinations may be employed tocreate a multi-transgenic animal model. Co-injection of differenttransgenes for generating multi-transgenic mice can be accomplished bythese and other methods routinely practiced in the art (see, e.g., Oddoet al., 2003, Neuron 39:409-421; U.S. Pat. No. 7,479,579).

Non-human animal models with senescent cell specific transgeneexpression created by cross-breeding, transgene injection, or othermethods may be confirmed for genotype or transgene expression. Resultingoffspring may be genotyped by Southern blot analysis or PCR techniqueson DNA extracted from tissue samples (e.g., tail tips or ear punches)using transgene specific probes or primers, respectively. The level ofmRNA expression of the transgenes in tissues of transgenic animals mayalso be assessed using techniques including Northern blot analysis, insitu hybridization, RT-PCR, or real-time PCR. Transgenic proteins mayalso be detected in tissue samples from transgenic animals usingantibodies specific for a polypeptide expressed by the transgene and/orantibodies that are specific for a detectable label that is co-expressedby the transgene, for example.

In another embodiment, for producing the animal model described herein,a knock out or knock-down animal (e.g., which develops an age-relatedphenotype of interest) may be crossed with a transgenic animal thatexpresses a senescent cell specific transgene. Gene knock-outs allowassessment of in vivo function of a gene which has been altered and usedto replace a normal copy. Knock-out modifications include insertion ofmutant stop codons, deletion of DNA sequences, or inclusion ofrecombination elements (lox p sites) recognized by enzymes such as Crerecombinase. Cre-lox system allows for the ablation of a given gene orthe ablation of a certain portion of gene sequence. To create atransgenic animal, an altered version of a gene of interest (e.g., ahuman age-related gene or a gene encoding a senescent cell associatedpolypeptide) can be inserted into an animal germ line using standardtechniques of oocyte microinjection or transfection, or microinjectioninto stem cells. For oocyte injection, one or more copies of thealtered/mutated gene of interest (e.g., human age-related gene) can beinserted into the pronucleus of a just-fertilized oocyte. The oocyte isthen re-implanted into a pseudo-pregnant foster mother. The livebornprogeny can be screened for transgene integrants by analyzing the DNAfrom tissue samples. Retroviral infection of early embryos may also beperformed to insert the altered gene. Embryos are infected during earlystages of development to generate a chimera, some of which will lead togermline transmission. Alternatively, if it is desired to inactivate orreplace the endogenous gene, mutant alleles may be introduced byhomologous recombination into embryonic stem cells. Embryonic stem cellscontaining a knock out mutation in one allele of the gene being studiedare introduced into early embryos. The resultant progeny are chimerascontaining tissues derived from both the transplanted ES cells and hostcells. Chimeric animals may be mated to assess whether the mutation isincorporated into the germ line. Chimeric animals that are eachheterozygous for the knock-out mutation are mated to produce homozygousknock out animals. Mutations in the mouse germline may also be createdby injecting oligonucleotides containing the mutation of interest. Geneknock down uses RNAi technology to repress endogenous gene expression invivo or in vitro. Lentiviral vectors expressing siRNAs or shRNAs may beused to transduce preimplantation mouse embryos for silencing of theirspecific target genes (see e.g., Tiscornia et al., 2003, Proc. Natl.Acad. Sci. USA 100:1844-1848; Singer et al., Nature Protocols 1:286-292;Szulc et al., 2006, Nature Methods 3:109-116).

Transgenic non-human animal models may comprise at least a secondtransgene associated with an age-related phenotype or disorder, inaddition to the first transgene selectively expressed by senescentcells. The expression of the second transgene will generally alter oneor more age-related phenotype or age-sensitive trait in the animal.

Gene knock out models for aging are known in the art and may compriseheterozygous or homozygous mutation of a gene associated with aging toreduce or abrogate expression of the gene. The reduction or abrogationof the gene associated with aging will generally result in orcontributes to one or more age-related phenotype or disorder, or willmodulate one or more age-sensitive traits.

Animal models of aging (including transgenic models, knockout models,and models having naturally occurring gene modifications) have beenreviewed (e.g., Exp Mol Pathol. 2002 February; 72(1):49-55; ILAR J. 2011Feb. 8; 52(1):4-15; and elsewhere, each of the review articles as wellas the references cited therein is incorporated herein by reference inits entirety).

Additionally, many transgenic/knockout mouse lines and information aboutmouse models of aging are available from the Jackson Laboratory (BarHarbor, Me.).

In certain embodiments, the present disclosure provides for an animalmodel comprising a p16^(Ink4a) promoter, or an operable truncationthereof, operatively linked to a polynucleotide sequence encoding aFKBP-caspase fusion polypeptide (p16-FKBP-caspase transgene), and to apolynucleotide sequence encoding a green fluorescent protein; or ap16^(Ink4a) promoter operatively linked to a polynucleotide sequenceencoding a fusion polypeptide comprising a luciferase, a red fluorescentprotein, and a truncated herpes simplex virus thymidine kinase (p16-3MRtransgene); wherein the model further comprises a second transgeneassociated with aging (e.g., one that gives rise to or contributes to anage-related phenotype, age-related disorder and/or age-sensitive trait).

In yet another embodiment, the present disclosure provides for a methodof producing a non-human animal model for aging comprising a non-humananimal that exhibits an age-related phenotype and comprises a transgeneselectively expressed by senescent cells, wherein the method comprises(a) providing an animal comprising the transgene selectively expressedby senescent cells; and (b) breeding the animal of step (a) with ananimal comprising a genetic modification associated with an age-relatedphenotype to produce a multi-transgenic animal. An animal comprising agenetic modification associated with an age-related phenotype may be aknockout, knockdown, or transgenic animal. In a specific embodiment, themethod may further comprise administering to, or exposing the animal to,for example, a chemical stimulus, whole body irradiation, anenvironmental stimulus, a diet modification, or another stimulus, toproduce or facilitate an age-related phenotype.

Non-human animal models for aging with senescent cell specific transgeneexpression, as described herein, are useful in tracking or monitoringsenescence cells. For example, animal models comprising transgenesexpressing detectable labels may be used in imaging senescent cells,determining ratio of senescent cells in a tissue, and/or monitoring theelimination of senescent cells. In addition, such models may also beused in characterizing drug candidates for treating or preventingdevelopment of an age-related disorder or age-sensitive trait, such asthe tissue specificity of such candidates. For example, these modelsexpressing detectable labels under the control of senescentcell-specific promoters may be used to determine the tissue type inwhich a drug candidate suppresses cellular senescence using thedetectable labels. Animal models comprising transgenes expressingcytotoxicity-activating molecules allow for titrating the elimination ofsenescent cells by modulating concentrations of activating agents. Inyet another embodiment, the present disclosure provides for a method ofproducing a non-human animal model for aging comprising a non-humananimal that exhibits an age-related phenotype and comprises a transgeneselectively expressed by senescent cells, wherein the method comprises(a) providing an animal comprising the transgene selectively expressedby senescent cells; and (b) administering a senescence-inducingstimulus.

Non-human animal models for aging with senescent cell specific transgeneexpression, as described herein, may be used to evaluate effects ofsenescent cell ablation on an age-related phenotype. By eliminatingsenescent cells or effects of senescent cell associated molecules fromthe animal at various times in an animal model for aging, the role ofsenescent cells in vivo on an age-related phenotype, particularly anage-related phenotype associated at least in part withsenescence-inducing stimuli, may be tested. In certain embodiments,senescent cell ablation may be accomplished by administration of FK1012analog AP20187, which induces dimerization of membrane-boundmyristolylated FKBP-caspase fusion protein expressed in senescent cellsvia the p16^(Ink4a) promoter, resulting in activation of apoptosis.Senescent cell ablation may also be performed by candidate therapeuticagents. In certain other embodiments, senescent cell ablation may beaccomplished by administration of the pro-drug ganciclovir (GCV), whichis converted to a cytotoxic moiety by a truncated herpes simplex virusthymidine kinase expressed in senescent cells via the p16^(Ink4a)promoter.

In certain embodiments, also provided herein are isolated cells and/orcell lines derived from the non-human animal models with senescentcell-specific transgene expression, as described herein. Primary cellcultures derived from the non-human animal models as described hereinmay be used. In certain embodiments, continuous cell lines are generatedfrom the non-human animal models. Methods for deriving a continuous cellline from transgenic animals are known in the art (see, e.g., Small etal., 1985, Mol. Cell. Biol. 5:642-648; Morgan et al., 1994, Dev. Biol.162:486-98; U.S. Pat. No. 5,814,716; U.S. Pat. No. 6,583,333; U.S. Pat.No. 6,825,394). Isolated cells or cell lines may be derived from anyorgan, tissue, or bodily fluid from the animal model, including, but notlimited to the brain, cerebrospinal fluid, or spinal cord. The cells andcell lines may be cultured under conditions and in media appropriate tomaintain the health and propagation of the cells, as desired, usingtechniques and procedures routinely practiced in the cell culture art.These isolated cells or cell lines may be used to identify andcharacterize therapeutic agents that suppress cellular senescence andthat are useful for treating or preventing age-related phenotypesassociated with senescence-inducing stimuli.

Methods for Identifying Therapeutic Agents

The non-human animal models and cell lines derived therefrom asdescribed herein may be used to identify therapeutic agents effectivefor treating or preventing age-related disorders, and, moreparticularly, for treating or preventing the deleterious age-relatedhealth effects caused by various senescence-inducing stimuli, such asradiation or chemotherapy treatment. Such animal models and cell linesare particularly useful for identifying therapeutic agents effective fortreating or preventing age-related disorders via suppressing cellularsenescence. Therapeutic agents include small molecules, antibodies,antigen-binding fragments, polypeptides, peptides, peptibodies,hormones, and nucleic acids.

In one embodiment, the present disclosure provides a method foridentifying a therapeutic agent effective for treating or preventing thedevelopment of an age-related disorder or age-sensitive trait,comprising: (a) administering a candidate therapeutic agent to theanimal of the animal model provided herein to provide a treated animal;and (b) determining the level of suppression of cellular senescence inthe treated animal and comparing to the level of cellular senescence inthe untreated control animal; wherein suppression of cellular senescencein the treated animal compared with the untreated animal identifies anagent effective for treating or preventing the age-related disorder orage-sensitive trait.

In a related embodiment, the present disclosure provides a method foridentifying a therapeutic agent effective for treating or preventing thedevelopment of an age-related disorder or age-sensitive trait,comprising: (a) administering a candidate therapeutic agent to theanimal of the animal model provided herein to provide a treated animal;and (b) (1) determining the phenotype of the development of anage-related disorder or age-sensitive trait exhibited in the treatedanimal, and comparing to the phenotype of the development of anage-related disorder or age-sensitive trait exhibited in an untreatedcontrol model animal, and (2) determining the level of suppression ofcellular senescence in the treated animal and comparing to the level ofcellular senescence in the untreated control animal; wherein (1)suppression of one or more age-related disorder or age-sensitive traitsand (2) suppression of cellular senescence in the treated animalcompared with the untreated animal identifies an agent effective fortreating or preventing the development of an age-related disorder orage-sensitive trait.

Candidate therapeutic agents include any agents that are potentiallycapable of treating or preventing one or more age-related disorder orage-sensitive trait, such as small molecules, antibodies, polypeptides,peptides, hormones, and nucleic acids. Potential therapeutic agents maybe identified from “libraries” or collections of compounds,compositions, or molecules. A source of small molecules, peptides, andoligonucleotides includes combinatorial libraries that may be screenedto identify a therapeutic agent useful for treating of preventing anage-related disorder or age-sensitive trait. Other exemplary librariescomprise peptides or polypeptides that represent a complementaritydetermining region (CDR) of an antibody.

Candidate therapeutic agents may be administered to the animal of theanimal model provided herein before, concurrently or after onset of anage-related disorder or age-sensitive trait. The onset of an age-relateddisorder or age-sensitive trait may be triggered by inducing theexpression of an inducible transgene that is associated with anage-related disorder or age-sensitive trait, such as by chemical ormechanical induction, by radiation, or by diet manipulation.

A person skilled in the art will also readily appreciate that whenperforming analyses in animal models, appropriate controls are included.By way of non-limiting example, a group of animals will receive acandidate therapeutic agent (or a composition or a vehicle comprisingthe agent), which animals may be referred to as treated animals. Anothergroup of animals will receive vehicle only or another appropriatecontrol composition that does not include an agent effective in treatingor preventing an age-related disorder or age-sensitive trait. In certainembodiments, a group of animals is treated with a agent known aseffective in treating or preventing an age-related disorder orage-sensitive trait as a positive control. The phenotype (or one or morephenotypic markers or characteristics) of the treated animal is thencompared with the phenotype of the control animals that do not receivethe candidate agent.

For these embodiments, a group of animals may receive a therapeuticagent known to be effective in treating or preventing an age-relateddisorder or age-sensitive trait by suppressing cellular senescence to beused as a positive control group. Additional positive controls includethe animal models provided herein that comprise a transgene thatincludes a senescent cell specific promoter that is operatively linkedto a polypeptide (which may be a fusion polypeptide) comprising acytotoxicity-activating molecule (e.g., HSV truncated TK, FKBP-caspasepolypeptide). Accordingly, when cells are induced to senescence bynormal aging or by any one of the molecules, methods, or geneticmodifications described herein, the fusion polypeptide expresses thecytotoxicity-activating molecule. By way of example, a positive controlanimal model group that expresses the FKBP-caspase polypeptide (e.g., anINK-ATTAC animal (see Baker et al., Nature 479:232-36 (2011)) may betreated with AP20187 and related analogs, for example, which results insenescent cell destruction. By way of additional example, a transgenicanimal (e.g., a P16-3MR animal) that expresses a fusion polypeptidecomprising HSV truncated TK can be administered a prodrug, such asganciclovir, that is activated when the truncated thymidine kinase isexpressed in senescent cells, resulting in destruction of the senescentcell.

Suppressing cellular senescence may comprise one or both of (1)selectively destroying or facilitating selective destruction of asenescent cell; and (2) inhibiting expression or secretion of one ormore senescence cell-associated molecules including senescence-cellassociated polypeptides (e.g., cytokines, chemokines, growth factors) bythe senescent cell.

Determining the effectiveness of a therapeutic agent or a candidatetherapeutic agent to inhibit induction or progression of an age-relateddisorder or age-sensitive trait or to suppress senescence as describedherein in an animal model is typically performed using one or morestatistical analyses with which a skilled person will be familiar. Byway of example, statistical analyses such as two-way analysis ofvariance (ANOVA) may be used for determining the statisticalsignificance of differences between animal groups treated with an agentand those that are not treated with the agent (i.e., negative controlgroup). Statistical packages such as SPSS, MINITAB, SAS, Statistika,Graphpad, GLIM, Genstat, and BMDP are readily available and routinelyused by a person skilled in the animal art.

Cellular senescence is a stable and essentially permanent arrest of cellproliferation, which is accompanied by extensive changes in geneexpression. Many types of cells, both normal cells and tumor cells,undergo senescence in response to stress. As described in the art, thephenotype of a senescence cell, such as the phenotype referred to assenescence associated secretory phenotype (SASP), is typified bysecretion of numerous cytokines (e.g., inflammatory cytokines), growthfactors, extracellular matrix components (ECM) and ECM-degradingenzymes, and proteases, for example. While proliferative arrest poses aformidable barrier to tumor progression (see, e.g., Campisi, Curr. Opin.Genet. Dev. 21:107-12 (2011); Campisi, Trends Cell Biol. 11:S27-31(2001); Prieur et al., Curr. Opin. Cell Biol. 20:150-55 (2008)), andmolecules secreted by senescent cells can stimulate tissue repair (see,e.g., Adams, Molec. Cell 36:2-14 (2009); Rodier et al., J. Cell Biol.192:547-56 (2011)), senescent cells also secrete molecules that cancause inflammation (see, e.g., Freund et al., Trends Mol. Med. 16:238-46(2010); Davalos et al., Cancer Metastasis Rev. 29:273-83 (2010)).Low-level, chronic inflammation is a hallmark of aging tissues, andinflammation is a major cause of, or contributor to, virtually everymajor age-related pathology, including cancer (Ferrucci et al., 2004,Aging Clin. Exp. Res. 16:240-243; Franceschi et al., 2007, Mech. AgeingDev. 128:192-105; Chung et al., 2009, Ageing Res. Rev. 8:18-30; Davaloset al., 2010, Cancer Metastasis Rev. 29:273-283; Freund et al., 2010,Trends Molec. Med. 16:238-248). Thus, senescent cells, which increasewith age and at sites of age-related pathology, might stimulate localchronic inflammation and tissue remodeling, thereby fueling both thedegenerative diseases of aging as well as age-related cancer.

A senescent cell may exhibit any one or more of the followingcharacteristics. (1) Senescence growth arrest is essentially permanentand cannot be reversed by known physiological stimuli. (2) Senescentcells increase in size, sometimes enlarging more than twofold relativeto the size of non-senescent counterparts. (3) Senescent cells express asenescence-associated β-galactosidase (SA-β-gal), which partly reflectsthe increase in lysosomal mass. (4) As described herein, most senescentcells express p16INK4a, which is not commonly expressed by quiescent orterminally differentiated cells. (5) Cells that senesce with persistentDNA damage response signaling harbor persistent nuclear foci, termed DNAsegments with chromatin alterations reinforcing senescence (DNA-SCARS).These foci contain activated DDR proteins and are distinguishable fromtransient damage foci. DNA-SCARS include dysfunctional telomeres ortelomere dysfunction-induced foci (TIF). (6) Senescent cells express andmay secrete molecules called herein senescent cell-associated molecules,which in certain instances may be dependent on persistent DDR signalingfor their expression. (7) The nuclei of senescent cells lose structuralproteins such as Lamin B1 or chromatin-associated proteins such ashistones and HMGB1. See, e.g., Freund et al., Mol. Biol. Cell 23:2066-75(2012); Davalos et al., J. Cell Biol. 201:613-29 (2013); Ivanov et al.,J. Cell Biol. DOI: 10.1083/jcb.201212110, page 1-15; published onlineJul. 1, 2013; Funayama et al., J. Cell Biol. 175:869-80 (2006)).

Senescent cell-associated molecules include growth factors, proteases,cytokines (e.g., inflammatory cytokines), chemokines, cell-relatedmetabolites, reactive oxygen species (e.g., H₂O₂), and other moleculesthat stimulate inflammation and/or other biological effects or reactionsthat may promote or exacerbate the underlying disease of the subject.Senescent cell-associated molecules include those that are described inthe art as comprising the senescence-associated secretory phenotype(SASP), senescent-messaging secretome, and DNA damage secretory program(DDSP). These groupings of senescent cell associated molecules, asdescribed in the art, contain molecules in common and are not intendedto describe three separate distinct groupings of molecules. Senescentcell-associated molecules include certain expressed and secreted growthfactors, proteases, cytokines, and other factors that may have potentautocrine and paracrine activities. Without wishing to be bound bytheory, the negative effects of senescent cells are believed to be theresult of, at least in part, the secretion of pro-inflammatorycytokines, chemokines, growth factors, and proteases that comprise theSASP of a senescent cell (see, e.g., Coppe et al., PLoS Biol. 6:2853-68(2008)). Senescent cell-associated molecules that comprise the SASP candisrupt normal tissue structure and function and stimulate malignantphenotypes in pre-malignant or non-aggressive cancer cells (see, e.g.,Coppe et al., supra; Coppe et al. J. Biol. Chem. 281:29568-74 (2006);Coppe et al. PLoS One 5:39188 (2010); Krtolica et al. Proc. Natl. Acad.Sci. U.S.A. 98:12072-77 (2001); Parrinello et al., J. Cell Sci.118:485-96 (2005). ECM associated factors include inflammatory proteinsand mediators of ECM remodeling and which are strongly induced insenescent cells (see, e.g., Kuilman et al., Nature Reviews 9:81-94(2009)). Other senescent cell-associated molecules include extracellularpolypeptides (proteins) described collectively as the DNA damagesecretory program (DDSP) (see, e.g., Sun et al., Nature Medicinepublished online 5 Aug. 2012; doi:10.1038/nm.2890). Senescentcell-associated proteins also include cell surface proteins (orreceptors) that are expressed on senescent cells, which include proteinsthat are present at a detectably lower amount or are not present on thecell surface of a non-senescent cell.

Senescence cell-associated molecules include secreted factors which maymake up the pro-inflammatory phenotype of a senescent cell (e.g., SASP).These factors include, without limitation, GM-CSF, GROα, GROα,β,γ,IGFBP-7, IL-1α, IL-6, IL-7, IL-8, MCP-1, MCP-2, MIP-1α, MMP-1, MMP-10,MMP-3, Amphiregulin, ENA-78, Eotaxin-3, GCP-2, GITR, HGF, ICAM-1,IGFBP-2, IGFBP-4, IGFBP-5, IGFBP-6, IL-13, IL-1β, MCP-4, MIF, MIP-3α,MMP-12, MMP-13, MMP-14, NAP2, Oncostatin M, osteoprotegerin, PIGF,RANTES, sgp130, TIMP-2, TRAIL-R3, Acrp30, angiogenin, Ax1, bFGF, BLC,BTC, CTACK, EGF-R, Fas, FGF-7, G-CSF, GDNF, HCC-4, I-309, IFN-γ,IGFBP-1, IGFBP-3, IL-1 R1, IL-11, IL-15, IL-2R-α, IL-6 R, I-TAC, Leptin,LIF, MMP-2, MSP-a, PAI-1, PAI-2, PDGF-BB, SCF, SDF-1, sTNF RI, sTNF RII,Thrombopoietin, TIMP-1, tPA, uPA, uPAR, VEGF, MCP-3, IGF-1, TGF-β3,MIP-1-delta, IL-4, FGF-7, PDGF-BB, IL-16, BMP-4, MDC, MCP-4, IL-10,TIMP-1, Fit-3 Ligand, ICAM-1, Ax1, CNTF, INF-γ, EGF, BMP-6.Cell-associated molecules also include those sometimes referred to inthe art as senescence messaging secretome (SMS) factors, some of whichare included in the listing of SASP polypeptides, include withoutlimitation, IGF1, IGF2, and IGF2R, IGFBP3, IDFBP5, IGFBP7, PA11, TGF-β,WNT2, IL-1α, IL-6, IL-8, and CXCR2-binding chemokines. Factors,including those referred to in the art, include without limitation thefactors described in Sun et al., Nature Medicine, supra, including, forexample, products of the genes, MMP1, WNT16B, SFRP2, MMP12, SPINK1,MMP10, ENPP5, EREG, BMP6, ANGPTL4, CSGALNACT, CCL26, AREG, ANGPT1, CCK,THBD, CXCL14, NOV, GAL, NPPC, FAM150B, CST1, GDNF, MUCL1, NPTX2,TMEM155, EDN1, PSG9, ADAMTS3, CD24, PPBP, CXCL3, MMP3, CST2, PSG8,PCOLCE2, PSG7, TNFSF15, C17orf67, CALCA, FGF18, IL8, BMP2, MATN3, TFP1,SERPINI 1, TNFRSF25, and IL23A. Senescent cell-associated proteins alsoinclude cell surface proteins (or receptors) that are expressed onsenescent cells, which include proteins that are present at a detectablylower amount or are not present on the cell surface of a non-senescentcell.

A therapeutic agent of interest includes an agent that selectivelydestroys or facilitates selective destruction of a senescent cell and/orin some manner is effective for inhibiting expression or secretion of asenescence cell-associated molecule, including a senescencecell-associated protein, or a protein that is present on the cellsurface of a senescent cell. Therapeutic agents of interest also includeagents that inhibit transcription or translation of a senescencecell-associated polypeptide (protein), or a protein that is present onthe cell surface of a senescent cell. Such agents are useful fortreating or preventing an age-related disorder or age-sensitive trait.

A therapeutic agent that “selectively” destroys or facilitates“selective” destruction of a senescent cell is an agent thatpreferentially (or to a greater degree) destroys or facilitatesdestruction or facilitates clearance of a senescent cell. In otherwords, the therapeutic agent destroys or facilitates destruction of asenescent cell in a biologically, clinically, and/or statisticallysignificant manner compared with its capability to destroy or facilitatedestruction of a non-senescent cell. By way of non-limiting example, thetherapeutic agent may directly or indirectly kill a senescent cell bydisrupting the integrity of the cell membrane; inhibiting one or moremetabolic processes in the cell; enhancing or stimulating a signalingpathway that leads to apoptosis or necrosis of the senescent cell;disrupt transcription or translation of genes or proteins, respectively,necessary for cell survival; and/or binding to the senescent cell tofacilitate clearance or removal of the cell, for example, clearance byimmune cells. As described herein, the presence of senescent cells inthe transgenic animals comprising a senescent cell specific promoter canbe monitored and determined by expression or presence (or lack ofexpression or presence) of one or more detectable labels (e.g., aluciferase or fluorescent polypeptide) that is operatively linked to thepromoter.

In particular embodiments, the level of transcription, expression, orsecretion can be determined for one or more senescence cell-associatedpolypeptides. An effective therapeutic agent that suppresses cellularsenescence reduces or inhibits expression, secretion, or production of asenescence cell-associated polypeptide in a statistically significant orbiologically significant manner compared to the appropriate controls.Proteins that comprise senescence cell-associated molecules and methodsfor evaluating expression and secretion of SASP proteins are describedin the art (see, e.g., Freund et al., Trends Mol. Med. 16:283-46 (2010)and references cited therein; Sun et al., Nature Med., published online5 Aug. 2012; doi: 10.1038/nm.2890). Senescent cells may also be detectedby determining the presence and level ofsenescence-associated-β-galactosidase (SA-β-Gal). A decrease orreduction in the level of expression or secretion of one or moresenescence cell-associated molecules (including senescencecell-associated polypeptides), SA-β-Gal, or reduction in the quantity ofsenescent cells and a suppression of one or more phenotypic markers ofan age-related disorder or age-sensitive trait in the test animalcompared with the control animal identifies a therapeutic agent.

Senescent cells and senescent cell associated molecules can be detectedby techniques and procedures described in the art. For example, thepresence of senescent cells in tissues can be analyzed by histochemistryor immunohistochemistry techniques that detect the senescence marker,SA-beta gal (SA-Bgal) (see, e.g., Dimri et al., Proc. Natl. Acad. Sci.USA 92: 9363-9367 (1995). The presence of the senescent cell-associatedpolypeptide p16 can be determined by any one of numerous immunochemistrymethods practiced in the art, such as immunoblotting analysis.Expression of p16 mRNA in a cell can be measured by a variety oftechniques practiced in the art including quantitative PCR. The presenceand level of senescence cell associated polypeptides (e.g., polypeptidesof the SASP) can be determined by using automated and high throughputassays, such as an automated Luminex array assay described in the art(see, e.g., Coppe et al., PLoS Biol 6: 2853-68 (2008)). For monitoring aDNA damage response, the various DNA damage response indicators can bedetected according to the method of Rodier et al., Nature Cell Biol 11:973-979 (2009)).

Therapeutic agents of interest include those that are activated or thatare pro-drugs which are converted to the active form by enzymes that areexpressed at a higher level in senescent cells than in non-senescentcells. Other therapeutic agents of interest include those that bind toproteins on the cell surface of a cell that are present exclusively orat a greater level on senescent cells compared with non-senescent cells(see, e.g., International Patent Application Publication No. WO2009/085216). In certain embodiments, a therapeutic agent thatspecifically binds to a senescent cell has at least 2, 4, 8, 10, 50,100, or 1000 fold greater affinity for a senescent cell than for anon-senescent cell, or in certain embodiments, the therapeutic agentdoes not detectably bind to a non-senescent cell. A protein present at agreater level on a senescent cell than on a non-senescent cell may be aprotein that is typically an intracellular protein and not detectable onthe cell surface of a non-senescent cell. Other therapeutic agents ofinterest that suppress cellular senescence include those that areactivated by a metabolic process that occurs more frequently or at ahigher rate in senescent cells than in a non-senescent cell.

In one embodiment, therapeutic agents useful for treating or preventingage-related disorder or an age-sensitive trait are small organicmolecules that suppress cellular senescence. A small molecule compoundof interest may be further derivatized, either randomly or by SAR, toobtain compounds with improved anti-cellular senescence activity andmore effective agents for treating an age-related disorder orage-sensitive trait. Small organic molecules typically have molecularweights less than 10⁵ daltons, less than 10⁴ daltons, or less than 10³daltons.

A therapeutic agent includes an antibody, or antigen-binding fragmentthereof, that specifically binds to a cognate antigen that is overlyexpressed, selectively expressed, or only expressed by senescent cellcompared with a non-senescent, normal cell. The antibody may be anantibody that is internalized by the senescent cell via interaction withits cognate antigen. These specific antibodies may be polyclonal ormonoclonal, prepared by immunization of animals and subsequent isolationof the antibody, or cloned from specific B cells according to methodsand techniques routinely practiced in the art and described herein. Avariable region or one or more complementarity determining regions(CDRs) may be identified and isolated from antigen-binding fragment orpeptide libraries. An antibody, or antigen-binding fragment thereof, maybe recombinantly engineered and/or recombinantly produced.

An antibody may belong to any immunoglobulin class, for example IgG,IgE, IgM, IgD, or IgA and may be obtained from or derived from ananimal, for example, fowl (e.g., chicken) and mammals, which include butare not limited to a mouse, rat, hamster, rabbit, or other rodent, acow, horse, sheep, goat, camel, human, or other primate. The antibodymay be an internalising antibody. For use in human subjects, antibodiesand antigen-binding fragments are typically human, humanized, orchimeric to reduce an immunogenic response by the subject to non-humanpeptides and polypeptide sequences.

Binding properties of an antibody to its cognate antigen may generallybe determined and assessed using immunodetection methods including, forexample, an enzyme-linked immunosorbent assay (ELISA),immunoprecipitation, immunoblotting, countercurrentimmunoelectrophoresis, radioimmunoassays, dot blot assays, inhibition orcompetition assays, and the like, which may be readily performed bythose having ordinary skill in the art (see, e.g., Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)).As used herein, an antibody is said to be “immunospecific,” “specificfor” or to “specifically bind” to a cognate antigen if it reacts at adetectable level with the antigen or immunogen. Affinities of antibodiesand antigen binding fragments thereof can be readily determined usingconventional techniques, for example, those described by Scatchard etal. (Ann. N.Y. Acad. Sci. USA 51:660 (1949)) and by surface plasmonresonance (SPR; BIAcore™, Biosensor, Piscataway, N.J.).

The antibody may be a monoclonal antibody that is a human antibody,humanized antibody, chimeric antibody, bispecific antibody, or anantigen-binding fragment (e.g., F(ab′)₂, Fab, Fab′, Fv, and Fd) preparedor derived therefrom. An antigen-binding fragment may also be anysynthetic or genetically engineered protein that acts like an antibodyin that it binds to a specific antigen to form a complex. For example,antibody fragments include isolated fragments consisting of the lightchain variable region, Fv fragments consisting of the variable regionsof the heavy and light chains, recombinant single chain polypeptidemolecules (scFv proteins), and minimal recognition units consisting ofthe amino acid residues that mimic the hypervariable region. In certainother embodiments, antibodies are multimeric antibody fragments such asminiantibodies, bispecific and bifunctional antibodies comprising afirst Fv specific for an antigen associated with a second Fv having adifferent antigen specificity, and diabodies and the like. Usefulmethodologies are described generally, for example in Hayden et al.,Curr Opin. Immunol. 9:201-12 (1997) and Coloma et al., Nat. Biotechnol.15:159-63 (1997); U.S. Pat. No. 5,910,573); (Holliger et al., CancerImmunol. Immunother. 45:128-30 (1997); Drakeman et al., Expert Opin.Investig. Drugs 6:1169-78 (1997); Koelemij et al., J. Immunother.22:514-24 (1999); Marvin et al., Acta Pharmacol. Sin. 26:649-58 (2005);Das et al., Methods Mol. Med. 109:329-46 (2005)).

A minimal recognition unit or other antigen binding fragment may beidentified from a peptide library. Such peptides may be identified andisolated from combinatorial libraries (see, e.g., International PatentApplication Nos. PCT/US91/08694 and PCT/US91/04666) and from phagedisplay peptide libraries (see, e.g., Scott et al., Science 249:386(1990); Devlin et al., Science 249:404 (1990); Cwirla et al., Science276: 1696-99 (1997); U.S. Pat. Nos. 5,223,409; 5,733,731; 5,498,530;5,432,018; 5,338,665; 1994; 5,922,545; International ApplicationPublication Nos. WO 96/40987 and WO 98/15833). A peptide that is aminimal recognition unit or a CDR (i.e., any one or more of three CDRspresent in a heavy chain variable region and/or one or more of threeCDRs present in a light chain variable region) may be identified bycomputer modeling techniques, which can be used for comparing andpredicting a peptide sequence that will specifically bind to apolypeptide of interest as described herein (see, e.g., Bradley et al.,Science 309:1868 (2005); Schueler-Furman et al., Science 310:638(2005)).

Antibodies may generally be prepared by any of a variety of techniquesknown to persons having ordinary skill in the art. Immunogens used toimmunize animals and/or to screen for antibodies of desired specificityinclude proteins isolated from senescent cells that, for example, arepresent on the cell surface of a senescent cell in greater quantity orhaving a different conformation than on a non-senescent cell; andsenescent cell extracts, including outer membrane preparations,organelles isolated from senescent cells, and the like. Antibodies mayalso be identified and isolated from human immunoglobulin phagelibraries, from rabbit immunoglobulin phage libraries, from mouseimmunoglobulin phage libraries, and/or from chicken immunoglobulin phagelibraries (see, e.g., Winter et al., Annu. Rev. Immunol. 12:433-55(1994); Burton et al., Adv. Immunol. 57:191-280 (1994); U.S. Pat. No.5,223,409; Huse et al., Science 246:1275-81 (1989); Schlebusch et al.,Hybridoma 16:47-52 (1997) and references cited therein; Rader et al., J.Biol. Chem. 275:13668-76 (2000); Popkov et al., J. Mol. Biol. 325:325-35(2003); Andris-Widhopf et al., J. Immunol. Methods 242:159-31(2000)).Antibodies isolated from non-human species or non-human immunoglobulinlibraries may be genetically engineered according to methods describedherein and known in the art to “humanize” the antibody or fragmentthereof.

Useful strategies for designing humanized antibodies may include, forexample by way of illustration and not limitation, identification ofhuman variable framework regions that are most homologous to thenon-human framework regions of a chimeric antibody (see, e.g., Jones etal., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-27(1988)). A humanized antibody may be designed to include CDR loopconformations and structural determinants of non-human variable regions,for example, by computer modeling, and then comparing the CDR loops anddeterminants to known human CDR loop structures and determinants (see,e.g., Padlan et al., FASEB 9:133-39 (1995); Chothia et al., Nature,342:377-83 (1989)). Computer modeling may also be used to compare humanstructural templates selected by sequence homology with the non-humanvariable regions.

A therapeutic agent also includes a peptide-immunoglobulin (Ig) constantregion fusion polypeptide, which includes a peptide-IgFc fusionpolypeptide. The peptide may be any naturally occurring or recombinantlyprepared molecule. A peptide-Ig constant region fusion polypeptide, suchas a peptide-IgFc fusion polypeptide (also referred to in the art as apeptibody (see, e.g., U.S. Pat. No. 6,660,843)).

Therapeutic agents such as polypeptides, peptides, peptibodies,antibodies, and antigen binding fragments (i.e., peptides orpolypeptides comprising at least one antibody V region) or other agentsthat specifically to a senescent cell can be linked to (i.e., conjugatedto, fused to, or in some manner joined to or attached to) a second agentthat selectively destroys or facilitates selective destruction ofsenescent cells. When delivered to the senescent cell by binding of theagent to the senescent cell, the cytotoxic moiety selectively destroysthe senescent cell. If the agent is recombinantly produced, a nucleotidesequence encoding the cytotoxic moiety may be linked in frame to theagent and to one or more regulatory expression sequences to produce afusion protein comprising the agent and cytotoxic moiety. Such secondagents include cytotoxic molecules, including toxins derived from plantsand microorganisms, as well as small molecules that do not selectivelybind to senescent cells in the absence of being linked to theaforementioned antibody, polypeptide, or peptide.

In certain embodiments, a therapeutic agent is a polynucleotide oroligonucleotide that specifically hybridize to a portion of the genomeor mRNA of a cell that is a senescent cell or that is in a diseasemicroenvironment and may be induced to senescence by a cell damaging(i.e., biologically damaging) medical therapy. Polynucleotides andoligonucleotides are provided that are complementary to at least aportion of a nucleotide sequence encoding a senescent cellularpolypeptide of interest (e.g., a short interfering nucleic acid, anantisense polynucleotide, a ribozyme, or a peptide nucleic acid) andthat may be used to alter gene and/or protein expression. As describedherein, these polynucleotides that specifically bind to or hybridize tonucleic acid molecules that encode a cellular polypeptide may beprepared using the nucleotide sequences available in the art. In anotherembodiment, nucleic acid molecules such as aptamers that are notsequence-specific may also be used to alter gene and/or proteinexpression.

Antisense polynucleotides bind in a sequence-specific manner to nucleicacids such as mRNA or DNA. Identification of oligonucleotides andribozymes for use as antisense agents and identification of DNA encodingthe genes for targeted delivery involve methods well known in the art.For example, the desirable properties, lengths, and othercharacteristics of such oligonucleotides are well known. Antisensetechnology can be used to control gene expression through interferencewith binding of polymerases, transcription factors, or other regulatorymolecules (see Gee et al., In Huber and Carr, Molecular and ImmunologicApproaches, Futura Publishing Co. (Mt. Kisco, N.Y.; 1994)).

Short interfering RNAs may be used for modulating (decreasing orinhibiting) the expression of a gene encoding a cellular polypeptide ofinterest. For example, small nucleic acid molecules, such as shortinterfering RNA (siRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules may be used according to the methods described hereinto modulate the expression of a cellular polypeptide of interest. AsiRNA polynucleotide preferably comprises a double-stranded RNA (dsRNA)but may comprise a single-stranded RNA (see, e.g., Martinez et al. Cell110:563-74 (2002)). A siRNA polynucleotide may comprise other naturallyoccurring, recombinant, or synthetic single-stranded or double-strandedpolymers of nucleotides (ribonucleotides or deoxyribonucleotides or acombination of both) and/or nucleotide analogues as provided herein andknown and used by persons skilled in the art.

In certain other embodiments, methods are provided herein foridentifying a therapeutic agent that suppresses cellular senescence byusing the animal models described herein. Candidate therapeutic agentsmay be administered to an animal of the animal model to provide atreated animal, followed by determining suppression of cellularsenescence as described herein (i.e., determining the level or extent towhich the candidate agent kills or facilitates killing of a senescentcell or determining the level of one or more senescence cell-associatedmolecules including senescence cell-associated polypeptides expressed orsecreted by a senescent cell). The capability of the agent to suppresscellular senescence is determined by comparing the level or extent towhich the candidate agent kills or facilitates killing of a senescentcell and/or comparing the level of one or more senescencecell-associated molecules such as senescence cell-associated proteinsexpressed or secreted by a senescent cell in the treated animal with anuntreated (i.e., vehicle only or placebo) control animal. Astatistically or biologically significant decrease or reduction ofcellular senescence in the treated animal compared with untreated,control animal thereby identifies an agent that suppresses cellularsenescence. As described herein, positive control animal groups (i.e.,those that include an agent capable of destroying or facilitatingdestruction of a senescent cell or that inhibit or reduce expression orsecretion of one or more senescence cell-associated including senescencecell-associated polypeptides) may also be included in such methods.

Also provided herein are methods for identifying a therapeutic agent fortreating and/or preventing age-related disorders and age-specific traitsassociated with senescence-inducing stimuli, which employ primary cellsfrom an animal model (e.g., an animal model mouse) or a cell lineprepared from cells isolated from the animal model. In one embodiment,primary cells or a cell line derived from the animal model may be usedin screening (including high throughput methods) for therapeutic agentsthat suppress cellular senescence. The cells may be exposed to,contacted, mixed with, or in some manner permitted to interact with anagent (e.g., a medical therapy) that induces cellular senescence priorto, concurrent with, or subsequent to contact with a candidatetherapeutic agent. Suppression of cellular senescence may be determinedby any of the methods described herein or in the art.

High throughput screening, typically automated screening, of a largenumber of candidate therapeutic agents from synthetic or natural productlibraries may be used to identify therapeutic agents. The candidatetherapeutic agents to be screened may be organized in a high throughputscreening format such as using microfluidics-based devices, or a 96-wellplate format, or other regular two dimensional array, such as a384-well, 48-well or 24-well plate format, or an array of test tubes.The format is therefore amenable to automation. An automated apparatusthat is under the control of a computer or other programmable controllermay be used for one or more steps of the methods described herein. Acontroller may monitor the results of each step of the method and mayautomatically alter the testing paradigm in response to those results.

Pharmaceutical Compositions and Methods of Treatment

The present disclosure further provides for pharmaceutical compositionscomprising any of the agents that suppress cellular senescenceidentified according to the methods described herein and apharmaceutically acceptable excipient. The therapeutic agents describedherein may be formulated in a pharmaceutical composition for use intreatment or preventive (or prophylactic) treatment (e.g., reducing thelikelihood of occurrence, of exacerbation of disease, or occurrence orrecurrence of one or more symptoms of the disease). The methods andexcipients described herein are exemplary and are in no way limiting.Pharmaceutical acceptable excipients are well known in thepharmaceutical art and described, for example, in Rowe et al., Handbookof Pharmaceutical Excipients: A Comprehensive Guide to Uses, Properties,and Safety, 5^(th) Ed., 2006, and in Remington: The Science and Practiceof Pharmacy (Gennaro, 21^(st) Ed. Mack Pub. Co., Easton, Pa. (2005)).Exemplary pharmaceutically acceptable excipients include sterile salineand phosphate buffered saline at physiological pH. Preservatives,stabilizers, dyes, buffers, and the like may be provided in thepharmaceutical composition. In addition, antioxidants and suspendingagents may also be used.

The pharmaceutical compositions may be in the form of a solution.Alternatively, they may be in the form of a solid, such as powder,tablets, or the like.

The present disclosure also provides a method for treating or preventingage-related disorders and age-sensitive traits associated withsenescence-inducing stimuli in a subject who has or who is at risk ofdeveloping an age-related disorder or age-sensitive trait associatedwith a senescence-inducing stimulus comprising administering atherapeutic agent that selectively suppresses cellular senescence in thesubject, thereby treating or preventing an age-related disorder orage-sensitive trait associated with a senescence-inducing stimulus inthe subject.

As understood by a person skilled in the medical art, the terms, “treat”and “treatment,” refer to medical management of a disease, disorder, orcondition of a subject (i.e., patient) (see, e.g., Stedman's MedicalDictionary). “Treating an age-related disorder or age-sensitive traitassociated with a senescence-inducing stimulus” refers to reducing thenumber of symptoms of an age-related disorder or age-sensitive traitassociated with a senescence-inducing stimulus, decreasing the severityof one or more symptoms, or delaying the progression of an age-relateddisorder or age-sensitive trait associated with a senescence-inducingstimulus.

“Preventing an age-related disorder or age-sensitive trait associatedwith a senescence-inducing stimulus” refers to preventing or delayingonset of an age-related disorder or age-sensitive trait associated witha senescence-inducing stimulus, or reoccurrence of one or moreage-related disorder or age-sensitive trait associated with asenescence-inducing stimulus.

Subjects at risk of developing an age-related disorder or age-sensitivetrait associated with a senescence-inducing stimulus include subjectsexposed to conditions which induce senescence. For example, in specificembodiments, subjects at risk include those exposed to asenescence-inducing stimulus, such as a chemical stimulus, anenvironmental stimulus, a genetic modification, a diet modification, ora combination thereof. In certain more specific embodiments, thesenescence-inducing stimulus comprises irradiation treatment (e.g.,whole body treatment) or treatment with one or more chemotherapeuticagents (e.g., doxorubicin, taxol, docetaxel, gemcitabine, cisplatin, andthe like). In other embodiments, the senescence-inducing stimuluscomprises cigarette smoking, or exposure to cigarette smoke or otherenvironmental insults.

The effectiveness of a therapeutic agent can readily be determined by aperson skilled in the medical and clinical arts. One or any combinationof diagnostic methods, including physical examination, assessment andmonitoring of clinical symptoms, and performance of analytical tests andmethods described herein, may be used for monitoring the health statusof the subject. The effects of the treatment of a therapeutic agent orpharmaceutical composition can be analyzed using techniques known in theart, such as comparing symptoms of patients suffering from or at risk ofan age-related disorder or age-sensitive trait that have received thetreatment with those of patients without such a treatment or withplacebo treatment.

In certain embodiments of the method for treating or preventing anage-related disorder or age-sensitive trait, the therapeutic agents areidentified according to the screening methods provided herein. Incertain other embodiments, the therapeutic agents may be other agentsknown in the art that selectively suppresses cellular senescence andthat treat and/or prevent an age-related disorder or age-sensitivetrait.

The therapeutic agents or pharmaceutical compositions that selectivelysuppress cellular senescence and that are useful for treating orpreventing an age-related disorder or age-sensitive trait may beadministered orally, topically, transdermally, parenterally, byinhalation spray, vaginally, rectally, or by intracranial injection, orany combination thereof. In one embodiment, the therapeutic agents orcompositions comprising the agents are administered parenterally, suchas via subcutaneous, intravenous, intramuscular, or intracisternalinjection, or via infusion techniques.

The therapeutic agents or pharmaceutical compositions that selectivelysuppress cellular senescence provided herein are administered to asubject who has or is at risk of developing an age-related disorder orage-sensitive trait associated with a senescence-inducing stimulus at atherapeutically effective dose. A “therapeutically effective dose” of aspecific therapeutic agent refers to that amount of the agent sufficientto result in reducing the severity of, eliminating, or delaying theonset or reoccurrence of one or more symptoms of an age-related disorderor age-sensitive trait in a statistically significant manner. Such adose may be determined or adjusted depending on various factorsincluding the specific therapeutic agents or pharmaceuticalcompositions, the routes of administration, the subject's condition,that is, stage of the disease, severity of symptoms caused by thedisease, general health status, as well as age, gender, and weight, andother factors apparent to a person skilled in the medical art.Similarly, the dose of the therapeutic for treating a disease ordisorder may be determined according to parameters understood by aperson skilled in the medical art. Optimal doses may generally bedetermined using experimental models and/or clinical trials. Design andexecution of pre-clinical and clinical studies for a therapeutic agent(including when administered for prophylactic benefit) described hereinare well within the skill of a person skilled in the relevant art. Theoptimal dose of a therapeutic may depend upon the body mass, weight, orblood volume of the subject. For example, an amount between 0.01 mg/kgand 1000 mg/kg (e.g., about 0.1 to 1 mg/kg, about 1 to 10 mg/kg, about10-50 mg/kg, about 50-100 mg/kg, about 100-500 mg/kg, or about 500-1000mg/kg) body weight (which can be administered as a single dose, daily,weekly, monthly, or at any appropriate interval) of a therapeutic agentmay be administered.

Also contemplated is the administration of a therapeutic agent or apharmaceutical composition that selectively suppresses cellularsenescence in combination with a second agent useful in treating orpreventing an age-related disorder or age-sensitive trait.

In certain embodiments, a therapeutic agent that selectively suppressescellular senescence and a second agent useful in treating or preventingan age-related disorder or age-sensitive trait act synergistically. Inother words, these two agents interact such that the combined effect ofthe agents is greater than the sum of the individual effects of eachagent when administered alone.

In certain other embodiments, a therapeutic agent that selectivelysuppresses cellular senescence and a second agent useful in treating orpreventing an age-related disorder or age-sensitive trait actadditively. In other words, these two agents interact such that thecombined effect of the agents is the same as the sum of the individualeffects of each agent when administered alone.

It is contemplated the therapeutic agent and the second agent may begiven simultaneously in the same formulation. Alternatively, the secondagents may be administered in a separate formulation but concurrently(i.e., given within less than one hour of each other).

In certain embodiments, the second agent useful in treating orpreventing an age-related disorder or age-sensitive trait may beadministered prior to administration of a therapeutic agent thatselectively suppresses cellular senescence. Prior administration refersto administration of the second agent at least one hour prior totreatment with the therapeutic agent. It is also contemplated that thesecond agent may be administered subsequent to administration of thetherapeutic agent. Subsequent administration is meant to describeadministration of the second agent at least one hour after theadministration of the therapeutic agent.

This disclosure contemplates a dosage unit comprising a pharmaceuticalcomposition provided herein. Such dosage units include, for example, asingle-dose or a multi-dose vial or syringe, including a two-compartmentvial or syringe, one comprising the pharmaceutical composition of thisdisclosure in lyophilized form and the other a diluent forreconstitution. A multi-dose dosage unit can also be, e.g., a bag ortube for connection to an intravenous infusion device.

The following examples are for illustration and are not limiting.

EXAMPLES Example 1 Preparation of P16-3MR Transgenic Mice

This Example describes preparation of a transgenic mouse comprising ap16^(ink4a) promoter operatively linked to a trimodal fusion protein.

The promoter, p16^(Ink4a), which is transcriptionally active insenescent cells but not in non-senescent cells (see, e.g., Wang et al.,J. Biol. Chem. 276:48655-61 (2001); Baker et al., Nature, supra) wasengineered into a nucleic acid construct. The p16^(Ink4a) gene promoter(approximately 100 kilobase pairs) was introduced upstream of anucleotide sequence encoding a trimodal reporter fusion protein.Alternatively, a truncated p16^(Ink4a) promoter may be used (see FIGS. 1and 2 providing an exemplary vector and exemplary promoter sequence)(see, e.g., Baker et al., Nature, supra; International ApplicationPublication No. WO/2012/177927; Wang et al., J. Biol. Chem. 276:48655-61(2001)). The trimodal reporter protein, the expression of which isdriven by the p16^(ink4a) promoter in senescent cells, is termed 3MR andconsists of renilla luciferase (rLUC), monomeric red fluorescent protein(mRFP) and a truncated herpes simplex virus thymidine kinase (tTK) (see,e.g., Ray et al., Cancer Res. 64:1323-30 (2004)). The polypeptidesequences and the encoding polynucleotides for each of the threeproteins are known in the art and are available in public databases,such as GenBank. An exemplary sequence (SEQ ID NO:25) for the 3MRtransgene is provided in FIG. 3. The 3MR transgene was inserted into aBAC vector using techniques routinely practiced by person skilled in themolecular biology art. The detectable markers, rLUC and mRFP permitteddetection of senescent cells by bioluminescence and fluorescence,respectively. The expression of tTK permitted selective killing ofsenescent cells by exposure to the pro-drug ganciclovir (GCV), which isconverted to a cytotoxic moiety by tTK. Transgenic founder animals,which have a C57B16 background, were established and bred according toroutinely procedures for introducing transgenes into animals (see, e.g.,Baker et al., Nature, supra). The transgenic mice are called p16-3MRherein.

Example 2 Effects of Therapy-Induced Senescence on Aging

This example identifies the role of senescence-inducing stimuli,particularly radiation and chemotherapy treatment, on declining healthparameters and age-sensitive traits using a p16-FKBP-caspase-8(INK-ATTAC) transgenic animal model. In particular, the exampledetermines the extent to which chemotherapy and gamma radiation inducecellular senescence, identifies that cellular senescence induced byvarious treatment regimens is deleterious for tissue structure andfunction, and identifies that clearance of therapy-induced senescentcells improves age-sensitive traits and other health parametersfollowing chemotherapy and radiation.

Briefly, INK-ATTAC mice are aged 10-12 weeks. Cohorts of mice aretreated with 4 Gy IR (4 cycles), doxorubicin (3-4 mg/kg i.p. daily forfive consecutive days: 3 cycles), docetaxel (25 mg/kg i.p. every sevendays for 3 cycles), or cisplatin (6 mg/kg i.p. every twenty days for 3cycles) to induce cellular senescence. The mice are also treated withAP20187 or PBS on a weekly basis from the start of treatment.

The rate of aging is assessed by various measures of health andlifespan. Specifically, mice in each of the above cohorts are analyzedfor the following age-sensitive traits, using methods known in the art,will not harm the animals, and do not require anesthesia: (1) T cellsubset distribution, (2) cataract formation, (3) spontaneous activity,(4) motor coordination, and (5) cognitive capacity. These traits provideassessments of systems known to change with age and which have importantimplications for human health. Spontaneous activity of individual miceis measured over a 48-hour period using comprehensive laboratory animalmonitoring systems equipped with photocells (e.g., ColumbusInstruments). Motor coordination is analyzed by performing theaccelerating rotarod test. For measuring cognitive capacity, a modifiedStone T-maze, which is sensitive to age-related changes in learning andmemory, is used.

Three additional age-sensitive traits known to change with age andhighly relevant to human health are also measured using knowntechniques: (1) physical function, (2) body composition (sarcopenia,osteoporosis, fat atrophy), and (3) cardiac function. Physical functionis assessed by measuring running time, distance, and work using amotorized treadmill, and grip strength using a grip meter. Lean mass,fat mass and bone mineral density is assessed by QNMR and/or dual-energyX-ray absorptiometry measurements.

The above analyses are complemented with assessments of age-sensitivetraits on tissues and organs of test and control mice. These analysesinclude the following assays which are performed using known techniques:(1) fiber diameter analysis on gastrocnemius muscle, (2) DNA damageanalysis, (3) analysis of renal and glomerulosclerosis, (4) analysis forretinal atrophy, (5) proteotoxic stress analysis, (6) oxidative stressanalysis, and (7) analysis of the hematopoietic system.

Example 3 Effects of Removing Senescent Cells from Aging Animal

Clearance of therapy-induced senescent cells improves age-sensitivetraits and other health parameters in aged animals. Wild type INK-ATTACtransgenic animals from each of lineage 3 (TG3) and lineage 5 (TG5) wereused in the experiments (see Baker et al., Nature, supra). INK-ATTACtransgenic mice were aged 10-12 weeks, and then cohorts of animals weretreated with AP20187 or PBS (control) on a weekly basis from the startof treatment. As shown in FIG. 4, animals treated with AP20187 had anaverage lifespan of approximately 26 months compared with 21 months forcontrol.

When the animals reached 18 months of age, several measures of healthand lifespan were taken. Weight, fat mass, activity measurements(treadmill exercise tests to measure distance traveled and duration ofexercise) were compared between untreated female mice (n=16) andAP20187-treated female mice (n=21), and compared between untreated malemice (n=19) and AP20187-treated male mice (n=21). Fat mass was greaterin both treated females and males compared with the untreated groups(see FIG. 5). No statistically significant differences were observedwith respect to weight, or activity measurements between the treated anduntreated animals. Treated and untreated mice (5-6 per group) weresacrificed, and adipose cell (AC) diameter, gastrocnemius muscle fiberdiameter (μm), abdominal muscle fiber diameter (μm), dermal thickness(μm), subdermal adipose thickness (μm), cardiac performance(introduction of isoproterenol as an exogenous stress in animals priorto sacrifice), and extent of glomerulosclerosis were determined. Treatedanimals had greater adipose cell diameter (see FIG. 6); better cardiacperformance (FIG. 7); and had less sclerotic glomeruli (see FIG. 8) thanuntreated animals. No significant differences between treated animalswere observed with respect to gastrocnemius muscle fiber diameter (μm),abdominal muscle fiber diameter (μm), dermal thickness (μm), andsubdermal adipose thickness (μm). Removal of senescent cells inAP20187-treated animals prevented loss of adipose tissue, prevented lossof cardiac performance, and reduced the extent of glomerulosclerosis.

The various embodiments described above can be combined to providefurther embodiments. All U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

We claim the following:
 1. A transgenic mouse for studying the role ofsenescent cells on an age-related disorder or an age-sensitive trait,wherein the genome of the mouse contains a recombinant polynucleotide,wherein the polynucleotide contains a p16^(INK4a) promoter sequence thatcontrols expression of an enzyme so as to cause the enzyme to beexpressed in senescent cells in the mouse; wherein the enzyme converts aprodrug to a cytotoxic agent; wherein treating the mouse with theprodrug results in the prodrug being converted to the cytotoxic agent bythe enzyme expressed from the recombinant polynucleotide in thesenescent cells, thereby killing the senescent cells but killing lessthan 10% of non-senescent cells; whereby progression of an age-relateddisorder or an age-sensitive trait is delayed in the mouse treated withthe prodrug compared with another such transgenic mouse that is nottreated with the prodrug.
 2. The transgenic mouse of claim 1, whereinthe p16^(INK4a) promoter also controls expression of a luminescentprotein so as to cause it to be expressed in senescent cells in themouse.
 3. The transgenic mouse of claim 1, wherein the p16^(INK4a)promoter also controls expression of a fluorescent protein so as tocause it to be expressed in senescent cells in the mouse.
 4. Thetransgenic mouse of claim 1, wherein the p16^(INK4a) promoter alsocontrols expression of a luminescent protein and a fluorescent proteinso as to cause the enzyme, the luminescent protein, and the fluorescentprotein all to be expressed in senescent cells in the mouse.
 5. Thetransgenic mouse of claim 1, wherein the enzyme is a thymidine kinase.6. The transgenic mouse of claim 1, wherein the prodrug is ganciclovir.7. The transgenic mouse of claim 2, wherein the luminescent protein isluciferase.
 8. The transgenic mouse of claim 3, wherein the fluorescentprotein is monomeric red fluorescent protein (mRFP).
 9. A mouse from astrain of mice having a transgene inserted in their genome, thetransgene containing: a p16^(INK4a) promoter sequence; a nucleotidesequence encoding an enzyme that converts a prodrug to a cytotoxicagent, wherein the enzyme is expressed under control of the p16^(INK4a)promoter so as to be produced by senescent cells in the mouse; and anucleotide sequence containing a luminescent and/or a fluorescent markerprotein, expressed under control of the p16^(INK4a) promoter so as to beproduced by senescent cells in the mouse; wherein treating the mousewith the prodrug results in the enzyme converting the prodrug to thecytotoxic agent in senescent cells, thereby killing the senescent cellsbut killing less than 10% of non-senescent cells; whereby progression ofan age-related disorder or an age-sensitive trait is delayed in themouse treated with the prodrug compared with an untreated mouse of thesame strain.
 10. The mouse of claim 9, wherein the enzyme and the markerprotein are expressed as a fusion protein.
 11. The mouse of claim 9,wherein the enzyme is a thymidine kinase and the prodrug is ganciclovir.12. The mouse of claim 9, wherein the marker protein is luciferase ormonomeric red fluorescent protein (mRFP).
 13. A method for determiningthe effect of a test compound on an age-related disorder or anage-sensitive trait mediated by senescent cells; the method comprising:obtaining a plurality of mice according to claim 1, dividing theplurality into several treatment groups, treating mice in a firsttreatment group with the prodrug, treating mice in a second treatmentgroup with the test compound, and imaging or measuring an effect of thetest compound on the age-related disorder or the age-sensitive trait inthe mice in the second treatment group.
 14. The method of claim 13,further comprising aging the mice or treating the mice with chemotherapyor radiation to promote the age-related disorder or the age-sensitivecondition.
 15. A method of visualizing senescent cells that mediate anage-related disorder or an age-sensitive trait, the method comprising:obtaining a mouse according to claim 9; promoting the age-relateddisorder or the age-sensitive trait in the mouse; and then imaging ormeasuring cells expressing the marker protein in the mouse.
 16. A methodfor determining the effect of a test compound on an age-related disorderor an age-sensitive trait mediated by senescent cells; the methodcomprising: obtaining a plurality of mice according to claim 9, dividingthe plurality into several treatment groups, treating mice in a firsttreatment group with the prodrug, treating mice in a second treatmentgroup with the test compound, and imaging or measuring an effect of thetest compound on the age-related disorder or the age-sensitive trait inthe mice in the second treatment group.
 17. A method for removingsenescent cells from a tissue, comprising contacting the senescent cellswith a prodrug, wherein the genome of cells in the tissue contains arecombinant polynucleotide in which a p16^(INK4a) promoter sequencecontrols expression of an enzyme so as to cause the enzyme to beexpressed in senescent cells; wherein the enzyme converts the prodrug toa cytotoxic agent; wherein contacting the senescent cells with theprodrug results in the prodrug being converted to the cytotoxic agent bythe enzyme expressed from the recombinant polynucleotide in thesenescent cells, thereby killing the senescent cells but killing lessthan 10% of non-senescent cells in the tissue; whereby progression of anage-related disorder or an age-sensitive trait is delayed in the tissueas a result of contacting the senescent cells in the tissue with theprodrug.
 18. The method of claim 17, wherein the p16^(INK4a) promoteralso controls expression of a luminescent protein so as to cause it tobe expressed in senescent cells in the tissue.
 19. The method of claim17, wherein the p16^(INK4a) promoter also controls expression of afluorescent protein so as to cause it to be expressed in senescent cellsin the method.
 20. The method of claim 17, wherein the enzyme is athymidine kinase and the prodrug is ganciclovir.